Calcium channel agonists

ABSTRACT

Embodiments of calcium channel agonists, as well as methods of making and using the calcium channel agonists, are disclosed. The disclosed calcium channel agonists and corresponding salt forms have a structure according to general formula I: 
                         
wherein each bond depicted as “ ” is a single bond or a double bond as needed to satisfy valence requirements; Z 1 , Z 2 , Z 3 , Z 4 , and Z 5  independently are nitrogen or carbon; R 1  and R 3  are alkyl; R 2  is alkyl, aryl, heteroaryl, arylalkyl, or heteroarylalkyl; and R 4  is alkyl or hydroxyalkyl.

RELATED APPLICATION

This application is the U.S. National Stage of International ApplicationNo. PCT/US2014/038574, filed May 19, 2014, which was published inEnglish under PCT Article 21(2), which in turn claims the benefit ofU.S. Provisional Application No. 61/825,392, filed May 20, 2013. Theprovisional application is incorporated herein by reference in itsentirety.

ACKNOWLEDGMENT OF GOVERNMENT SUPPORT

This invention was made with government support under Grant NumberGM067082 awarded by the National Institutes of Health and Grant Number0844604 awarded by National Science Foundation. The government hascertain rights in the invention.

FIELD

This disclosure concerns embodiments of calcium channel agonists, aswell as methods of making and using the calcium channel agonists.

BACKGROUND

Chemical communication in the nervous system is tightly regulated by theflux of calcium ions through certain subtypes of voltage-gated channels.A decrease in calcium flux at synapses can cause neurological diseases.For example, Lambert-Eaton Myasthenic Syndrome (LEMS) is a neurologicalautoimmune disorder of the neuromuscular junction characterized bydebilitating muscle weakness. While LEMS is often a paraneoplasticsyndrome associated with small cell lung cancer, it can also beidiopathic. This muscle weakness has been shown to be due to anauto-antibody-mediated removal of a fraction of presynaptic P/Q-type(Cav2.1) calcium channels, which are known to be involved withtransmitter release at the mammalian neuromuscular junction, and apartial compensatory up-regulation of N-type (Cav2.2), L-type (Cav1),and R-type (Cav2.3) channels. N-type and P/Q-type channels appear to bethe most relevant for the control of transmitter release as theyselectively bind directly to and co-localize with transmitter releasesites. Despite a compensatory expression of other calcium channel types,the overall effect is a decrease in the quantal content of transmitterrelease from the NMJ. LEMS results in muscle weakness and is associatedwith compromised motor function. This disease is estimated to affect1:100,000 individuals in the United States; however, the true incidenceof LEMS remains unknown as it is often undiagnosed in patients.

Current treatment strategies are very limited, and those available areindirect and sometimes associated with undesirable side effects. Ifcancer is present, anti-tumor therapy is the priority. In any case, thistype of neuromuscular weakness can be treated using eitherimmunosuppressants or symptomatic treatment approaches.Immunosuppressants have not been favored, as side-effects may be severeand include leukopenia, liver dysfunction, nausea, vomiting, and hairloss. The most common therapeutic approach is the use of the potassiumchannel blocker 3,4-diaminopyridine (DAP), which indirectly increasespresynaptic Ca²⁺ entry by broadening the action potential waveform,leading to an increase in transmitter release. In clinical trials, 10-20mg of the potassium channel blocker 3,4-diaminopyridine (DAP), whichincreases calcium entry by broadening the action potentialdepolarization, was given 3 times per day, and led to serum levels ofabout 0.5 μM. However, DAP is only partially effective in LEMS. Althoughgenerally well-tolerated, DAP can have dose-limiting side-effects thatinclude paresthesia, gastric symptoms, difficulty in sleeping, fatigue,and deterioration of muscle. The latter two may be due to reportedeffects on axonal K⁺ channels that limit firing frequencies and/orreduction in activity-dependent facilitation caused by DAP. Thus, thecurrent LEMS treatment approach indirectly increases calcium entry intothe nerve terminal, but there are currently no other common treatmentoptions.

Another disorder in which calcium channel agonists may be useful ismyasthenia gravis. Myasthenia gravis is an autoimmune disordercharacterized by blockade or loss of acetylcholine receptors at theneuromuscular junction. The symptoms of this disorder are often managedusing acetylcholinesterase blockers.

Selective calcium channel agonists which increase the ion flux throughN- and P/Q-type calcium channels represent attractive potentialtherapeutics for LEMS and other neuromuscular diseases; however, todate, no such agonists have been identified.

SUMMARY

Embodiments of calcium channel agonists, and pharmaceutical compositionsincluding calcium channel agonists, are disclosed. Methods of making andusing the calcium channel agonists also are disclosed. Embodiments ofthe disclosed compounds have a structure according to general formula Ior a pharmaceutically acceptable salt thereof:

wherein each bond depicted as “

” is a single bond or a double bond as needed to satisfy valencerequirements; Z¹, Z², Z³, Z⁴, and Z⁵ independently are nitrogen orcarbon; R¹ and R³ are alkyl, such as C₁-C₃ alkyl; R² is alkyl, aryl,heteroaryl, arylalkyl, or heteroarylalkyl; and R⁴ is alkyl orhydroxyalkyl, provided that: when Z¹ and Z³ are nitrogen, Z², Z⁴, and Z⁵are carbon, R¹ is 2-propyl, R³ is ethyl, and R⁴ is —CH₂OH, then R² isnot benzyl or 2-hydroxybenzyl.

In some embodiments, two of Z¹, Z², Z³, Z⁴, and Z⁵ are nitrogen. Incertain embodiments, Z¹ and Z³ are nitrogen, and Z², Z⁴, and Z⁵ arecarbon.

In some embodiments, R¹ is n-alkyl. In one embodiment, R¹ is n-propyl.In some embodiments, R² is arylalkyl or heteroarylalkyl. In oneembodiment, R² is substituted or unsubstituted thiophenyl methyl. In oneembodiment, R³ is ethyl. In another embodiment, R⁴ is —CH₂OH.

One exemplary compound has the structure:

Embodiments of a pharmaceutical composition comprising a calcium channelagonist comprise at least one compound according to general formula I ora pharmaceutically acceptable salt thereof, and at least onepharmaceutically acceptable additive.

Embodiments of a method for treating condition mediated by calciumchannel dysfunction include administering to a subject having, orsuspected of having, a condition mediated by calcium channel dysfunctiona therapeutically effective amount of a according to general formula Ior a pharmaceutically acceptable salt thereof. Conditions mediated bycalcium channel dysfunction include conditions that produceneuromuscular weakness. Exemplary conditions include Lambert-Eatonmyasthenic syndrome, congenital myasthenic syndrome, myasthenia gravis(e.g., MuSK myasthenia gravis), botulism, botulinum toxin overdose, aperipheral demyelinating disorder (e.g., Guillain-Barré syndrome,chronic inflammatory demyelinating polyneuropathy, anti-MAG peripheralneuropathy, Charcot-Marie-Tooth disease, copper deficiency), a motorneuron disease (e.g., spinal muscular atrophy, amyotrophic lateralsclerosis, primary lateral sclerosis, progressive bulbar palsy,pseudobulbar palsy), or a combination thereof.

In some embodiments, the compound has a Ca²⁺ channel activity halfmaximal effective concentration, EC₅₀, of ≦50 μM. The compound may havea cyclin-dependent kinase 2 EC₅₀ of at least 0.2 μM. In certainembodiments, the compound has an N-type and/or P/Q-type Ca²⁺ channelactivity EC₅₀ that is at least 10-fold less than an L-type Ca²⁺ channelactivity EC₅₀ of the compound.

Embodiments of the disclosed method may further include administering tothe subject a therapeutically effective amount of anacetylcholinesterase inhibitor, an immunosuppressant, intravenousimmunoglobulins, a glucocorticoid, ascorbic acid, an anti-cancer agent,a potassium channel blocker, a copper supplement, an analgesic, anantidepressant, a muscle relaxant, or a combination thereof. When thecondition is Lambert-Eaton myasthenic syndrome, the method may furtherinclude administering to the subject a therapeutically effective amountof 3,4-diaminopyridine.

The foregoing and other objects, features, and advantages of theinvention will become more apparent from the following detaileddescription, which proceeds with reference to the accompanying figures.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is the molecular structure of (R)-roscovitine.

FIG. 2 is a scheme illustrating a synthetic strategy used to makeselective Ca²⁺ channel agonists.

FIG. 3A shows the chemical structures of (R)-roscovitine and 3 analogs:compounds 13u, 13w, 13x. Circles indicate structural differencescompared to (R)-roscovitine.

FIG. 3B is a series of Ca²+ channel agonist activity dose-responsecurves for each of the compounds shown in FIG. 3A: (R)-roscovitine (♦),13u (▪), 13w (●), and 13x (▴). Each analog-modified tail currentintegral was normalized to its peak tail current and then divided by itsrespective control (untreated) tail current integral (also normalized toits respective peak current) to calculate the final value. Error barsindicate standard error of the mean (s.e.m.).

FIG. 3C illustrates representative tail current traces for each of thecompounds, along with a control tail current trace for comparison.

FIG. 4A is a plot of cdk activity measured at varying concentrations of(R)-roscovitine and compound 13x. Compound 13x inhibits cdk2 at morethan 20-fold higher concentration than (R)-roscovitine.

FIG. 4B illustrates calcium tail current (bottom traces) evoked by arepolarizing voltage step (top trace). Under control conditions (cont;no drugs), calcium current decays very quickly. After exposure to(R)-roscovitine (Ros), decay of calcium current is significantly slowed.Exposure to a newly developed analog of (R)-roscovitine (13x) slowscalcium current deactivation more dramatically than (R)-roscovitine.

FIG. 4C is a dose response curve for (R)-roscovitine (Ros) and compound13x demonstrating that 13x has a 6-fold higher affinity for the calciumchannel than (R)-roscovitine.

FIG. 5 illustrates molecular docking of compounds 13d, 13g, 13k, 13w,13x and 13u to the cdk2/roscovitine complex. The electrostaticinteraction surface at the binding site region is displayed and coloredred for negative charge and blue for positive charge. Dockingsimulations were performed using Molegro Virtual Docker, taking intoaccount side chain flexibility for all residues in the binding region.

FIG. 6 illustrates hydrogen bond interactions between compounds 13d,13g, 13k, 13u, 13w, 13x and cdk2. Interacting chemical groups of theanalogs are shown in blue ellipses on the (R)-roscovitine scaffold.

FIG. 7A is a graph showing an evaluation of several patients' serumevaluated in a LEMS passive transfer model by measuring quantal contentfollowing the passive transfer protocol. White bars indicate asignificant decrease in quantal content compared to controlserum-treated NMJs, while black bars indicate no significant differencefrom control serum-treated NMJs.

FIG. 7B is a graph showing an evaluation of the serum samples of FIG. 7Afor levels of voltage-gated Ca²+ channel (VGCC) antibodies.

FIG. 7C illustrates sample mEPP (miniature end plate potential, insets)and EPP (end plate potential) traces from a representative control (leftpanel) and aBC2 serum-treated (right panel) NMJ. Treatment with aBC2serum resulted in a decrease in EPP amplitude, but no change in averagemEPP amplitude. Error bars indicate s.e.m.

FIG. 8A shows sample traces (overlay of 10 traces in each example)showing the increase in EPP amplitude following a 30-minute incubationin 50 μM 13x relative to vehicle control (0.05% DMSO).

FIG. 8B is an average of 10 traces from the same NMJ before (vehicle)and after 30-minute incubation in 50 μM 13x show a 13x-induced wideningof the EPP trace.

FIG. 8C shows representative mEPP traces from the same NMJ before andafter 13x application.

FIG. 8D illustrates that the quantal content determined by measuring thepeak (peak EPP amplitude divided by the average peak mEPP amplitude) wasslightly, but significantly smaller following 50 μM oloumucine (Olom.)application, but was significantly increased following 13x application.The scatter plot represents the variability between individual synapsesstudied. Error bars indicate s.e.m.

FIG. 8E shows that the quantal content determined by measuring the area(EPP area divided by average mEPP area) was not significantly differentfollowing 50 μM oloumucine (Olom.) application, but was significantlyincreased following 13x application. The scatter plot represents thevariability between individual synapses studied. Error bars indicates.e.m.

FIG. 9A shows representative EPPs evoked by 50 Hz stimuli recorded fromterminals in control serum-injected mice and aBC2 LEMS serum-injectedmice.

FIG. 9B is a plot of the average 50 Hz train data normalized to theamplitude of the first EPP of the train for the two conditions shown inFIG. 9A. Error bars indicate s.e.m.

FIG. 9C shows representative 50 Hz trains for aBC2 LEMS serum-injectedmice in the presence of the DMSO vehicle and aBC2 LEMS serum-injectedmice following application of 50 μM 13x.

FIG. 9D is a plot of the average 50 Hz train data normalized to theamplitude of the first EPP of the train for the two conditions shown inFIG. 9C. Error bars indicate s.e.m.

FIGS. 10A and 10B. The synergistic effect of GV-58 plus DAP completelyreverses the deficit in neurotransmitter release at LEMS model NMJs.FIG. 10A—Sample traces showing the average EPP amplitudes followingexposure to a variety of conditions. Left: sample average EPP recordedfrom a NMJ of a control serum-treated mouse. Middle: sample average EPPsof a LEMS model NMJ before and after application of 50 μM GV58 (alsoreferred to herein as compound 13x). Right: sample average EPPs recordedfrom a LEMS model NMJ before drug application (LEMS), followingapplication of 1.5 μM DAP, and following application of 50 μM GV-58 plus1.5 μM DAP. FIG. 10B Plot of the quantal content for NMJs in each of thefive conditions: NMJs from control serum-treated mice (n=41), LEMS modelNMJs in the presence of the vehicle (n=63), LEMS model NMJs followingapplication of 50 μM GV-58 (n=20), LEMS model NMJs following applicationof 1.5 μM DAP (n=21) and LEMS model NMJs following application of 50 μMGV-58 plus 1.5 μM DAP (n=63). Data are represented as mean±s.e.m.

FIGS. 11A and 11B. The synergistic effect of GV-58 plus DAP elicits anear complete restoration of short-term synaptic plasticitycharacteristics in LEMS model NMJs. FIG. 11A Sample EPPs recorded fromNMJs in each of the five conditions during a 50 Hz train of 10 stimuli.Dashed lines indicate the amplitude of the first EPP in each train. FIG.11B Plot of the average change in EPP amplitude during a 50 Hz stimulustrain for NMJs from control serum-treated mice (n=41), LEMS model NMJsin vehicle (n=75), LEMS model NMJs after application of 50 μM GV-58(n=24), LEMS model NMJs following application of 1.5 μM DAP (n=21), andLEMS model NMJs following application of 50 μM GV-58 plus 1.5 μM DAP(n=75). Each EPP in the train is first normalized to the first EPP inthe train before averaging responses from many trials. The averagenormalized values are then plotted for each treatment condition. Dataare represented as mean±s.e.m. Dashed line represents no change from theamplitude of the first EPP in the train.

FIG. 12 is a graph of data regarding cell toxicity for compound 13x.

FIG. 13 is a graph of data regarding the dose-response effect on calciumchannels of compound MF-521-17 (also referred to herein as compound 9).

DETAILED DESCRIPTION

Embodiments of calcium channel agonists are disclosed. Methods of makingand using the calcium channel agonists also are disclosed.

The disclosed compounds are analogs of (R)-roscovitine. Some embodimentsof the disclosed compounds have an increased agonist effect for Ca²⁺channels, have a more potent effect on Ca²⁺ current, and also exhibitreduced kinase (e.g., cyclin-dependent kinase) activity compared to(R)-roscovitine. Certain embodiments of the disclosed calcium channelagonists target presynaptic Ca²⁺ channels and can partially restore thedeficiency of transmitter release in a LEMS passive transfer mouse modelneuromuscular junction.

I. Terms and Abbreviations

The following explanations of terms and abbreviations are provided tobetter describe the present disclosure and to guide those of ordinaryskill in the art in the practice of the present disclosure. As usedherein, “comprising” means “including” and the singular forms “a” or“an” or “the” include plural references unless the context clearlydictates otherwise. The term “or” refers to a single element of statedalternative elements or a combination of two or more elements, unlessthe context clearly indicates otherwise.

Unless explained otherwise, all technical and scientific terms usedherein have the same meaning as commonly understood to one of ordinaryskill in the art to which this disclosure belongs. Although methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present disclosure, suitable methods andmaterials are described below. The materials, methods, and examples areillustrative only and not intended to be limiting. Other features of thedisclosure are apparent from the following detailed description and theclaims.

Unless otherwise indicated, all numbers expressing quantities ofreactants and products, properties such as molecular weight,percentages, dosages, and so forth, as used in the specification orclaims are to be understood as being modified by the term “about.”Accordingly, unless otherwise indicated, implicitly or explicitly, thenumerical parameters and/or non-numerical properties set forth areapproximations that may depend on the desired properties sought, limitsof detection under standard test conditions/methods, limitations of theprocessing method, and/or the nature of the parameter or property. Whendirectly and explicitly distinguishing embodiments from discussed priorart, the embodiment numbers are not approximates unless the word “about”is recited.

Definitions of common terms in chemistry may be found in Richard J.Lewis, Sr. (ed.), Hawley's Condensed Chemical Dictionary, published byJohn Wiley & Sons, Inc., 1997 (ISBN 0-471-29205-2). Definitions ofcommon terms in molecular biology may be found in Benjamin Lewin, GenesVII, published by Oxford University Press, 2000 (ISBN 019879276X);Kendrew et al. (eds.), The Encyclopedia of Molecular Biology, publishedby Blackwell Publishers, 1994 (ISBN 0632021829); and Robert A. Meyers(ed.), Molecular Biology and Biotechnology: a Comprehensive DeskReference, published by Wiley, John & Sons, Inc., 1995 (ISBN0471186341); and other similar references.

In order to facilitate review of the various embodiments of thedisclosure, the following explanations of specific terms are provided:

Agonist: A compound that binds to a receptor or an enzyme and producesan action. For example, an agonist that binds to a cellular receptorinitiates a physiological or pharmacological response characteristic ofthat receptor. An agonist that binds to an enzyme activates the enzyme.An antagonist blocks an action of an agonist.

Alkyl: A hydrocarbon group having a saturated carbon chain. The chainmay be cyclic, branched or unbranched. The term lower alkyl means thechain includes 1-10 carbon atoms. Unless otherwise stated, the alkylgroup may be substituted or unsubstituted.

Aralkyl or arylalkyl: An aryl group (such as a phenyl group) appended toan alkyl radical including, but not limited to, benzyl, ethylbenzene,propylbenzene, butylbenzene, pentylbenzene, and the like. Conversely theterm “phenylalkyl” refers to a phenyl group appended to an alkylradical. Aralkyl groups, such as benzyl groups, may be unsubstituted orsubstituted with one, two or three substituents, with substituent(s)independently selected from alkyl, heteroalkyl, aliphatic,heteroaliphatic, thioalkoxy, haloalkyl (such as —CF₃), halo, nitro,cyano, —OR (where R is hydrogen or alkyl), —N(R)R′ (where R and R′ areindependently of each other hydrogen or alkyl), —COOR (where R ishydrogen or alkyl) or —C(O)N(R′)R″ (where R′ and R″ are independentlyselected from hydrogen or alkyl). Non-limiting examples, include o-, m-,and/or p-chlorobenzyl, o-, m-, and/or p-methoxybenzyl, and o-, m-,and/or p-(trifluoromethyl)benzyl. Unless otherwise stated, the aralkylor arylalkyl group may be substituted or unsubstituted.

Aryl: A monovalent aromatic carbocyclic group of, unless specifiedotherwise, from 6 to 15 carbon atoms having a single ring (e.g., phenyl,isoxazole) or multiple condensed rings which condensed rings may or maynot be aromatic (e.g., quinolone, indole, benzodioxole, and the like),provided that the point of attachment is through an atom of an aromaticportion of the aryl group and the aromatic portion at the point ofattachment contains only carbons in the aromatic ring. If any aromaticring portion contains a heteroatom, the group is a heteroaryl and not anaryl. Aryl groups are monocyclic, bicyclic, tricyclic or tetracyclic.Unless otherwise stated, the aryl group may be substituted orunsubstituted.

Calcium channel: An ion channel, which is selectively permeable tocalcium ions. Voltage-dependent calcium channels (VDCCs) are found inmembranes of certain cells, such as muscle cells and neurons. A VDCC isnormally closed. When activated, or opened, the VDCC allows Ca²⁺ toenter the cell, thereby resulting in, e.g., muscular contraction,neuronal excitation, or neurotransmitter release, depending on the celltype.

Cycloalkyl: A saturated monovalent cyclic hydrocarbon radical of threeto seven ring carbons, e.g., cyclopentyl, cyclohexyl, cycloheptyl andthe like. Unless otherwise stated, the cycloalkyl group may besubstituted or unsubstituted.

Effective amount or therapeutically effective dose: An amount sufficientto provide a beneficial, or therapeutic, effect to a subject or a givenpercentage of subjects.

Heteroaryl: An aromatic compound or group having at least oneheteroatom, i.e., one or more carbon atoms in the ring has been replacedwith an atom having at least one lone pair of electrons, typicallynitrogen, oxygen, phosphorus, silicon, or sulfur. Unless otherwisestated, the heteroaryl group may be substituted or unsubstituted.

Heteroarylalkyl: An arylalkyl radical as defined above containing atleast one heteroatom in the aryl group, typically nitrogen, oxygen,phosphorus, silicon, or sulfur. Unless otherwise stated, theheteroarylalkyl group may be substituted or unsubstituted.

Hydroxyalkyl: An alkyl radical substituted with at least one hydroxylgroup. In some examples, a hydroxyalkyl group has the general formula—ROH where R is alkyl, such as lower alkyl. Unless otherwise stated, Rmay be substituted or unsubstituted. Exemplary hydroxyalkyl groupsinclude hydroxymethyl (—CH₂OH) and hydroxyethyl (—CH₂CH₂OH).

Pharmaceutically acceptable salt: A biologically compatible salt of acompound that can be used as a drug, which salts are derived from avariety of organic and inorganic counter ions well known in the art.

Selectivity: As used herein, selectivity refers to the ability of acompound to preferentially affect activity of a particular calciumchannel and/or kinase. For example, the compound may preferentiallyactivate N and/or P/Q-type over L-type calcium channels.

Stereochemistry: The relative spatial arrangement of atoms that form thestructure of a molecule. Compounds with the same molecular formula andsequence of bonded atoms, but which differ only in the three-dimensionalorientation of the atoms in space, are called stereoisomers.

Subject: An animal or human subjected to a treatment, observation orexperiment.

Substituent: An atom or group of atoms that replaces another atom in amolecule as the result of a reaction. The term “substituent” typicallyrefers to an atom or group of atoms that replaces a hydrogen atom on aparent hydrocarbon chain or ring.

Substituted: A fundamental compound, such as an aryl or alkyl compound,or a radical thereof, having coupled thereto, typically in place of ahydrogen atom, a second substituent. For example, substituted arylcompounds or substituents may have an aliphatic group coupled to theclosed ring of the aryl base, such as with toluene. Again solely by wayof example and without limitation, a hydrocarbon may have a substituentbonded thereto, such as one or more halogens, an aryl group, a cyclicgroup, a heteroaryl group or a heterocyclic group.

Treating or treatment: With respect to disease, either term includes (1)preventing the disease, e.g., causing the clinical symptoms of thedisease not to develop in an animal that may be exposed to orpredisposed to the disease but does not yet experience or displaysymptoms of the disease, (2) inhibiting the disease, e.g., arresting thedevelopment of the disease or its clinical symptoms, or (3) relievingthe disease, e.g., causing regression of the disease or its clinicalsymptoms.

II. Calcium Channel Agonists

(R)-Roscovitine (FIG. 1) is a trisubstituted purine that was originallydeveloped as a cyclin-dependent kinase (cdk) inhibitor. Cdks have beenimplicated in neuronal development, synaptic transmission, cytoskeletalcontrol, neurodegeneration, and cell cycle control. In terms of clinicaluse, some inhibitors of cdks are being tested for use as anti-cancerdrugs and in the treatment of neurodegenerative diseases. Roscovitine isa chiral compound, and both (R) and (S) configurations are effective cdkinhibitors. The phenyl ring, isopropyl group, and ethyl group attachedto stereogenic carbon of roscovitine are involved in hydrophobicinteractions within the ATP binding site of cdk. (R)-roscovitine is alsoa potent agonist for a subset of voltage-gated calcium channels, i.e.,N- and P/Q-type calcium channels. These channel subtypes are the twomajor subtypes that regulate chemical transmitter release in the nervoussystem. (R)-Roscovitine slows the deactivation kinetics of N- andP/Q-type Ca²⁺ channels by increasing their mean open time, which leadsto an increase in transmitter release at synapses. Although(R)-roscovitine does target the Ca²⁺ channels involved in transmitterrelease at the NMJ, the potent (R)-roscovitine-mediated inhibition ofcdks presents a potential source of undesirable side-effects if used forthe treatment of LEMS. The development of a selective agonist for N- andP/Q-type calcium channels could have a significant impact on thetreatment of LEMS as well as other neurological diseases.

At the single channel level, it is known that calcium channels normallygate with a short (predominant) or long (rare) mean open time. It hasbeen shown that (R)-roscovitine significantly prolongs the mean opentime of calcium channels gating with a long open time, and increases theprobability of observing channels that gate with a long open time. Theseeffects lead to increased calcium flux when channels are naturallyactivated by an action potential, which increases transmitter release atneuromuscular and CNS synapses.

Embodiments of (R)-roscovitine analogs and correspondingpharmaceutically acceptable salts are disclosed. In some embodiments,the compounds are selective voltage-gated calcium channel agonists,e.g., N- and/or P/Q-type calcium channel agonists. Certain embodimentsof the disclosed compounds slow deactivation (closing) of the calciumchannel, resulting in increased calcium entry during motor nerve actionpotential activity. The compounds also may have decreased kinaseactivity, such as decreased cyclin-dependent kinase (cdk) activity.

Desirably, (R)-roscovitine analogs would exhibit 10-100 times reducedcdk activity and stronger, 10-100 times higher affinity calcium channelagonist effects that would be appropriate to treat neuromuscularweakness mediated by calcium channel dysfunction. A 4-zone approach(FIG. 1) was used for initial medicinal chemistry structure activityrelationship (SAR) studies. Zone 1 modifications indicated that someminor structural changes were not deleterious to calcium channelactivity. In zone 2, calcium channel agonist activity was more potentwhen the nitrogen was substituted with a hydrogen atom; modifications tothe aryl ring were permitted. When the stereochemistry in zone 3 wasaltered, the desired Ca²⁺ channel agonist activity was lost. In zone 4,some modifications were acceptable.

The potential side-effects of a use-dependent N- and P/Q-type calciumchannel agonist that enhances calcium flux through calcium channels thatare normally activated by action potential activity are expected to befew and manageable. This expectation is based on an extensive clinicalliterature that uses an indirect method of achieving a similar outcomewith potassium channel blockers such as 3,4-diaminopyridine (DAP). DAPprolongs the duration of the pre-synaptic action potential and thisindirectly increases the activation of all voltage-gated calciumchannels in the peripheral nervous system. DAP is generally welltolerated by LEMS patients, with many of the side-effects reportedlikely due to either broadening of action potentials in all neurons orthe non-selective indirect effect of increasing calcium flux through allvoltage-gated channels. Based on these previous reports, selectiveuse-dependent N- and P/Q-type calcium channel agonists are predicted tobe well tolerated.

Embodiments of the disclosed calcium channel agonists and correspondingsalt forms have a structure and stereochemistry according to generalformula I:

wherein each bond depicted as “

” is a single bond or a double bond as needed to satisfy valencerequirements; Z¹, Z², Z³, Z⁴, and Z⁵ independently are nitrogen orcarbon; R¹ and R³ are alkyl; R² is alkyl, cycloalkyl, aryl, heteroaryl,arylalkyl, or heteroarylalkyl; and R⁴ is alkyl or hydroxyalkyl, providedthat when Z¹ and Z³ are nitrogen, Z², Z⁴, and Z⁵ are carbon, R¹ is2-propyl, R³ is ethyl, and R⁴ is —CH₂OH, then R² is not benzyl or2-hydroxybenzyl.

In some embodiments, two of Z¹, Z², Z³, Z⁴, and Z⁵ are nitrogen. In oneembodiment, Z¹ and Z³ are nitrogen, and Z², Z⁴, and Z⁵ are carbon. Inanother embodiment, Z¹ and Z² are nitrogen, and Z³, Z⁴, and Z⁵ arecarbon. In yet another embodiment, Z¹ and Z⁵ are nitrogen, and Z², Z³,and Z⁴ are carbon.

In some embodiments, R¹ is n-alkyl, such as lower n-alkyl. In certainexamples, R¹ is C₁-C₃ alkyl, e.g., methyl, ethyl, n-propyl, or 2-propyl.In some embodiments, R² is arylalkyl or heteroarylalkyl. In certainexamples, R² is heteroarylalkyl, e.g., substituted or unsubstitutedthiophenyl methyl.

In some embodiments, R³ is lower alkyl. In one embodiment, R³ is ethyl.In certain embodiments, R⁴ is hydroxyalkyl, such as —R⁵OH, where R⁵ islower alkyl, for example, C₁-C₃ alkyl. In one embodiment, R⁴ ishydroxymethyl.

In some embodiments, the calcium channel agonist is a pharmaceuticallyacceptable salt form of a compound according to general formula I.Suitable salts may include sodium salts, potassium salts, argininesalts, choline salts, calcium salts, and pharmaceutically acceptableacid or base addition salts, but generally any pharmaceuticallyacceptable salt may be used for methods described herein. Generally,pharmaceutically acceptable salts are those salts that retainsubstantially one or more of the desired pharmacological activities ofthe parent compound and which are suitable for administration to humans.

Exemplary compounds 13a-13x according to formula I are shown in Table 1.In each of compounds 13a-13x, Z¹ and Z³ are nitrogen, Z², Z⁴, and Z⁵ arecarbon, R³ is ethyl, and R⁴ is —CH₂OH.

TABLE 1 Compound R¹ R² (R)-roscovitine i-Pr Bn 13a Pr CH₂(biphenyl) 13bPr CH(Ph)₂ 13c Pr (CH₂)₂Ph 13d Pr Bn 13e Pr Ph 13f Pr CH₂(3-Py) 13g PrCH₂CH(CH₂)₂ 13h Me CH₂(biphenyl) 13i Me CH(Ph)₂ 13j Me (CH₂)₂Ph 13k MeBn 13l Me Ph 13m Me CH₂CH(CH2)₂ 13n Me CH₂(3-Py) 13o PrCH₂[(p-trifluoromethyl)phenyl] 13p i-Pr CH₂CH(CH₂)₂ 13q i-Pr (CH₂)₂Ph13r i-Pr CH₂(3-Py) 13s i-Pr CH₂(biphenyl) 13t i-Pr CH(Ph)₂ 13u i-PrCH₂[(2-methyl)5-thiophenyl] 13v i-PrCH₂[(m-trifluoromethyl)5-thiophenyl] 13w Me CH₂[(2-methyl)5-thiophenyl]13x Pr CH₂[(2-methyl)5-thiophenyl] i-Pr = isopropyl, Pr = propyl, Me =methyl, Bn = benzyl, Ph = phenyl, Py = pyridineIII. Compound Synthesis

A synthetic strategy useful for making some embodiments of selectiveCa²⁺ channel agonists is summarized in FIG. 2. Starting with thecommercially available 2,6-dichloropurine 14, N-9 alkylation of thepurine with primary alkyl halides leads to intermediate 15. PreferentialS_(N)Ar reaction at the more reactive C-6 position followed bydisplacement of the C-2 chloride with primary amines provides targetanalogs 13.

Zone 4 modifications are achieved by subjecting dichloropurine 14 to adeprotonation in dimethyl sulfoxide (DMSO) in the presence of a mildbase such as potassium carbonate, followed by the addition of alkylbromides or iodides at 16-18° C. Primary alkyl halides (R¹=Me, Pr)provide a similar yield (64-78%) to the secondary alkyl halide (R¹=i-Pr,65%). Microwave irradiation at 120° C. for 20 minutes in n-butanol andtriethylamine with various aryl- and alkylamines converts intermediates15 to the C-6 aminated purines 16 in moderate-to-good yields (52-95%).Finally, aminolysis at C-2 under forcing conditions (e.g., heating neatin a sealed flask at 170° C. in the presence of the R³-amine for 8-15hours) introduces an (R)-2-amino-1-butanol side chain to give targetmolecules 13 in variable yields (25-93%).⁵

IV. Methods of Use

A. Treatment of Conditions Mediated by Ca²⁺ Channel Dysfunction

Embodiments of the disclosed calcium channel agonists are useful fortreating conditions mediated by calcium channel dysfunction, includingconditions that produce neuromuscular weakness. Exemplary conditionsinclude, but are not limited to Lambert-Eaton Myasthenic Syndrome(LEMS), congenital myasthenic syndrome (a heterogeneous group ofinherited disorders caused by mutations in any one of >10 genes thatcode for synaptic proteins, leading to impaired neuromuscular function),myasthenia gravis (e.g., MuSK myasthenia gravis—characterized byantibodies against the MuSK protein (muscle specific kinase), a tyrosinekinase receptor required for formation of neuromuscular junctions),botulism, botulinum toxin overdose (e.g., from onabotulinumtoxin Ainjections (Botox) for therapeutic and cosmetic purposes), a peripheraldemyelinating disorder (e.g., Guillain-Barré syndrome, chronicinflammatory demyelinating polyneuropathy, anti-MAG peripheralneuropathy, Charcot-Marie-Tooth disease, copper deficiency), a motorneuron disease (e.g., spinal muscular atrophy, amyotrophic lateralsclerosis, primary lateral sclerosis, progressive bulbar palsy,pseudobulbar palsy), or a combination thereof.

In some embodiments, an effective amount of a compound according togeneral formula I or a pharmaceutically acceptable salt thereof isadministered to a subject having, or suspected of having, a conditionmediated by calcium channel dysfunction, or a subject for whom a calciumchannel agonist would improve the symptoms resulting from a neurologicdisorder not mediated by calcium channel dysfunction.

The calcium channel agonists described herein, or compositions thereof,will generally be used in an amount effective to achieve the intendedresult, for example in an amount effective to treat a condition mediatedby calcium channel dysfunction. By therapeutic benefit is meanteradication or amelioration of one or more of the symptoms associatedwith the underlying disorder such that the subject reports animprovement in feeling or condition, notwithstanding that the subjectmay still be afflicted with the underlying disorder.

The amount of compound administered will depend upon a variety offactors, including, for example, the particular condition being treated,the mode of administration, the severity of the condition being treatedand the age and weight of the patient, the bioavailability of theparticular active compound, etc. Determination of an effective dosage iswell within the capabilities of those skilled in the art. A skilledpractitioner will be able to determine the optimal dose for a particularindividual. Effective dosages may be estimated initially from in vitroor in vivo assays. For example, an initial dosage for use in animals maybe formulated to achieve a circulating blood or serum concentration ofactive compound that is at or above an EC₅₀ of the particular compoundas measured in an in vitro assay or an in vivo assay as described belowin Example 3. Calculating dosages to achieve such circulating blood orserum concentrations taking into account the bioavailability of theparticular compound is well within the capabilities of skilled artisans.For guidance, the reader is referred to Fingl & Woodbury, “GeneralPrinciples,” In: Goodman and Gilman's The Pharmaceutical Basis ofTherapeutics, latest edition, Pergamon Press, and the references citedtherein.

Embodiments of the disclosed calcium channel agonists may beadministered by oral, parenteral (for example, intramuscular,intraperitoneal, intravenous, ICV, intracisternal injection or infusion,subcutaneous injection, or implant), by inhalation spray, nasal,vaginal, rectal, sublingual, urethral (for example, urethralsuppository) or topical routes of administration (for example, gel,ointment, cream, aerosol, etc.) and may be formulated, alone ortogether, in suitable dosage unit formulations containing conventionalnon-toxic pharmaceutically acceptable carriers, adjuvants, excipientsand vehicles appropriate for each route of administration. In additionto the treatment of warm-blooded animals such as mice, rats, horses,cattle, sheep, dogs, cats, monkeys, etc., the compounds described hereinmay be effective in humans.

Pharmaceutical compositions for administration to a subject can includeat least one further pharmaceutically acceptable additive such ascarriers, thickeners, diluents, buffers, preservatives, surface activeagents and the like in addition to the calcium channel agonist.Pharmaceutical compositions can also include one or more additionalactive ingredients such as antimicrobial agents, anti-inflammatoryagents, anesthetics, and the like. The pharmaceutically acceptablecarriers useful for these formulations are conventional. Remington'sPharmaceutical Sciences, by E. W. Martin, Mack Publishing Co., Easton,Pa., 19th Edition (1995), describes compositions and formulationssuitable for pharmaceutical delivery of the compounds disclosed herein.

In general, the nature of the carrier will depend on the particular modeof administration being employed. For instance, parenteral formulationsusually contain injectable fluids that include pharmaceutically andphysiologically acceptable fluids such as water, physiological saline,balanced salt solutions, aqueous dextrose, glycerol or the like as avehicle. For solid compositions (for example, powder, pill, tablet, orcapsule forms), conventional non-toxic solid carriers can include, forexample, pharmaceutical grades of mannitol, lactose, starch, ormagnesium stearate. In addition to biologically-neutral carriers,pharmaceutical compositions to be administered can contain minor amountsof non-toxic auxiliary substances, such as wetting or emulsifyingagents, preservatives, and pH buffering agents and the like, for examplesodium acetate or sorbitan monolaurate.

Pharmaceutical compositions disclosed herein include those formed frompharmaceutically acceptable salts and/or solvates of the disclosedcalcium channel agonists. Pharmaceutically acceptable salts includethose derived from pharmaceutically acceptable inorganic or organicbases and acids. Particular disclosed calcium channel agonists possessat least one basic group that can form acid-base salts with acids.Examples of basic groups include, but are not limited to, amino andimino groups. Examples of inorganic acids that can form salts with suchbasic groups include, but are not limited to, mineral acids such ashydrochloric acid, hydrobromic acid, sulfuric acid or phosphoric acid.Basic groups also can form salts with organic carboxylic acids, sulfonicacids, sulfo acids or phospho acids or N-substituted sulfamic acid, forexample acetic acid, propionic acid, glycolic acid, succinic acid,maleic acid, hydroxymaleic acid, methylmaleic acid, fumaric acid, malicacid, tartaric acid, gluconic acid, glucaric acid, glucuronic acid,citric acid, benzoic acid, cinnamic acid, mandelic acid, salicylic acid,4-aminosalicylic acid, 2-phenoxybenzoic acid, 2-acetoxybenzoic acid,embonic acid, nicotinic acid or isonicotinic acid, and, in addition,with amino acids, for example with α-amino acids, and also withmethanesulfonic acid, ethanesulfonic acid, 2-hydroxymethanesulfonicacid, ethane-1,2-disulfonic acid, benzenedisulfonic acid,4-methylbenzenesulfonic acid, naphthalene-2-sulfonic acid, 2- or3-phosphoglycerate, glucose-6-phosphate or N-cyclohexylsulfamic acid(with formation of the cyclamates) or with other acidic organiccompounds, such as ascorbic acid. In particular, suitable salts includethose derived from alkali metals such as potassium and sodium, alkalineearth metals such as calcium and magnesium, among numerous other acidswell known in the pharmaceutical art.

Certain calcium channel agonists may include at least one acidic groupthat can form an acid-base salt with an inorganic or organic base.Examples of salts formed from inorganic bases include salts of thepresently disclosed compounds with alkali metals such as potassium andsodium, alkaline earth metals, including calcium and magnesium and thelike. Similarly, salts of acidic compounds with an organic base, such asan amine (as used herein terms that refer to amines should be understoodto include their conjugate acids unless the context clearly indicatesthat the free amine is intended) are contemplated, including saltsformed with basic amino acids, aliphatic amines, heterocyclic amines,aromatic amines, pyridines, guanidines and amidines. Of the aliphaticamines, the acyclic aliphatic amines, and cyclic and acyclic di- andtri-alkyl amines are particularly suitable for use in the disclosedcompounds. In addition, quaternary ammonium counterions also can beused.

Particular examples of suitable amine bases (and their correspondingammonium ions) for use in the present calcium channel agonists include,without limitation, pyridine, N,N-dimethylaminopyridine,diazabicyclononane, diazabicycloundecene, N-methyl-N-ethylamine,diethylamine, triethylamine, diisopropylethylamine, mono-, bis- ortris-(2-hydroxyethyl)amine, 2-hydroxy-tert-butylamine,tris(hydroxymethyl)methylamine, N,N-dimethyl-N-(2-hydroxyethyl)amine,tri-(2-hydroxyethyl)amine and N-methyl-D-glucamine. For additionalexamples of “pharmacologically acceptable salts,” see Berge et al., J.Pharm. Sci. 66:1 (1977). The pharmaceutical compositions can beadministered to subjects by a variety of mucosal administration modes,including by oral, rectal, intranasal, intrapulmonary, or transdermaldelivery, or by topical delivery to other surfaces. Optionally, thecompositions can be administered by non-mucosal routes, including byintramuscular, subcutaneous, intravenous, intra-arterial,intra-articular, intraperitoneal, intrathecal, intracerebroventricular,or parenteral routes. In other alternative embodiments, the compound canbe administered ex vivo by direct exposure to cells, tissues or organsoriginating from a subject.

To formulate the pharmaceutical compositions, the calcium channelagonist can be combined with various pharmaceutically acceptableadditives, as well as a base or vehicle for dispersion of the compound.Desired additives include, but are not limited to, pH control agents,such as arginine, sodium hydroxide, glycine, hydrochloric acid, citricacid, and the like. In addition, local anesthetics (for example, benzylalcohol), isotonizing agents (for example, sodium chloride, mannitol,sorbitol), adsorption inhibitors (for example, Tween 80 or Miglyol 812),solubility enhancing agents (for example, cyclodextrins and derivativesthereof), stabilizers (for example, serum albumin), and reducing agents(for example, glutathione) can be included. Adjuvants, such as aluminumhydroxide (for example, Amphogel, Wyeth Laboratories, Madison, N.J.),Freund's adjuvant, MPL™ (3-O-deacylated monophosphoryl lipid A; Corixa,Hamilton, Ind.) and IL-12 (Genetics Institute, Cambridge, Mass.), amongmany other suitable adjuvants well known in the art, can be included inthe compositions. When the composition is a liquid, the tonicity of theformulation, as measured with reference to the tonicity of 0.9% (w/v)physiological saline solution taken as unity, is typically adjusted to avalue at which no substantial, irreversible tissue damage will beinduced at the site of administration. Generally, the tonicity of thesolution is adjusted to a value of about 0.3 to about 3.0, such as about0.5 to about 2.0, or about 0.8 to about 1.7.

The calcium channel agonist can be dispersed in a base or vehicle, whichcan include a hydrophilic compound having a capacity to disperse thecompound, and any desired additives. The base can be selected from awide range of suitable compounds, including but not limited to,copolymers of polycarboxylic acids or salts thereof, carboxylicanhydrides (for example, maleic anhydride) with other monomers (forexample, methyl (meth)acrylate, acrylic acid and the like), hydrophilicvinyl polymers, such as polyvinyl acetate, polyvinyl alcohol,polyvinylpyrrolidone, cellulose derivatives, such ashydroxymethylcellulose, hydroxypropylcellulose and the like, and naturalpolymers, such as chitosan, collagen, sodium alginate, gelatin,hyaluronic acid, and nontoxic metal salts thereof. Often, abiodegradable polymer is selected as a base or vehicle, for example,polylactic acid, poly(lactic acid-glycolic acid) copolymer,polyhydroxybutyric acid, poly(hydroxybutyric acid-glycolic acid)copolymer and mixtures thereof. Alternatively or additionally, syntheticfatty acid esters such as polyglycerin fatty acid esters, sucrose fattyacid esters and the like can be employed as vehicles. Hydrophilicpolymers and other vehicles can be used alone or in combination, andenhanced structural integrity can be imparted to the vehicle by partialcrystallization, ionic bonding, cross-linking and the like. The vehiclecan be provided in a variety of forms, including fluid or viscoussolutions, gels, pastes, powders, microspheres and films for directapplication to a mucosal surface.

The calcium channel agonist can be combined with the base or vehicleaccording to a variety of methods, and release of the compound can be bydiffusion, disintegration of the vehicle, or associated formation ofwater channels. In some circumstances, the calcium channel agonist isdispersed in microcapsules (microspheres) or nanocapsules (nanospheres)prepared from a suitable polymer, for example, isobutyl 2-cyanoacrylate(see, for example, Michael et al., J. Pharmacy Pharmacol. 43:1-5, 1991),and dispersed in a biocompatible dispersing medium, which yieldssustained delivery and biological activity over a protracted time.

The compositions of the disclosure can alternatively contain aspharmaceutically acceptable vehicles substances as required toapproximate physiological conditions, such as pH adjusting and bufferingagents, tonicity adjusting agents, wetting agents and the like, forexample, sodium acetate, sodium lactate, sodium chloride, potassiumchloride, calcium chloride, sorbitan monolaurate, and triethanolamineoleate. For solid compositions, conventional nontoxic pharmaceuticallyacceptable vehicles can be used which include, for example,pharmaceutical grades of mannitol, lactose, starch, magnesium stearate,sodium saccharin, talcum, cellulose, glucose, sucrose, magnesiumcarbonate, and the like.

Pharmaceutical compositions for administering the calcium channelagonist can also be formulated as a solution, microemulsion, or otherordered structure suitable for high concentration of active ingredients.The vehicle can be a solvent or dispersion medium containing, forexample, water, ethanol, polyol (for example, glycerol, propyleneglycol, liquid polyethylene glycol, and the like), and suitable mixturesthereof. Proper fluidity for solutions can be maintained, for example,by the use of a coating such as lecithin, by the maintenance of adesired particle size in the case of dispersible formulations, and bythe use of surfactants. In many cases, it will be desirable to includeisotonic agents, for example, sugars, polyalcohols, such as mannitol andsorbitol, or sodium chloride in the composition. Prolonged absorption ofthe calcium channel agonist can be brought about by including in thecomposition an agent which delays absorption, for example, monostearatesalts and gelatin.

In certain embodiments, the calcium channel agonist can be administeredin a time release formulation, for example in a composition whichincludes a slow release polymer. These compositions can be prepared withvehicles that will protect against rapid release, for example acontrolled release vehicle such as a polymer, microencapsulated deliverysystem or bioadhesive gel. Prolonged delivery in various compositions ofthe disclosure can be brought about by including in the compositionagents that delay absorption, for example, aluminum monostearatehydrogels and gelatin. When controlled release formulations are desired,controlled release binders suitable for use in accordance with thedisclosure include any biocompatible controlled release material whichis inert to the active agent and which is capable of incorporating thecalcium channel agonist and/or other biologically active agent. Numeroussuch materials are known in the art. Useful controlled-release bindersare materials that are metabolized slowly under physiological conditionsfollowing their delivery (for example, at a mucosal surface, or in thepresence of bodily fluids). Appropriate binders include, but are notlimited to, biocompatible polymers and copolymers well known in the artfor use in sustained release formulations. Such biocompatible compoundsare non-toxic and inert to surrounding tissues, and do not triggersignificant adverse side effects, such as nasal irritation, immuneresponse, inflammation, or the like. They are metabolized into metabolicproducts that are also biocompatible and easily eliminated from thebody.

Exemplary polymeric materials for use in the present disclosure include,but are not limited to, polymeric matrices derived from copolymeric andhomopolymeric polyesters having hydrolyzable ester linkages. A number ofthese are known in the art to be biodegradable and to lead todegradation products having no or low toxicity. Exemplary polymersinclude polyglycolic acids and polylactic acids, poly(DL-lacticacid-co-glycolic acid), poly(D-lactic acid-co-glycolic acid), andpoly(L-lactic acid-co-glycolic acid). Other useful biodegradable orbioerodable polymers include, but are not limited to, such polymers aspoly(epsilon-caprolactone), poly(epsilon-caprolactone-CO-lactic acid),poly(epsilon.-caprolactone-CO-glycolic acid), poly(beta-hydroxy butyricacid), poly(alkyl-2-cyanoacrilate), hydrogels, such as poly(hydroxyethylmethacrylate), polyamides, poly(amino acids) (for example, L-leucine,glutamic acid, L-aspartic acid and the like), poly(ester urea),poly(2-hydroxyethyl DL-aspartamide), polyacetal polymers,polyorthoesters, polycarbonate, polymaleamides, polysaccharides, andcopolymers thereof. Many methods for preparing such formulations arewell known to those skilled in the art (see, for example, Sustained andControlled Release Drug Delivery Systems, J. R. Robinson, ed., MarcelDekker, Inc., New York, 1978). Other useful formulations includecontrolled-release microcapsules (U.S. Pat. Nos. 4,652,441 and4,917,893), lactic acid-glycolic acid copolymers useful in makingmicrocapsules and other formulations (U.S. Pat. Nos. 4,677,191 and4,728,721) and sustained-release compositions for water-soluble peptides(U.S. Pat. No. 4,675,189).

The pharmaceutical compositions of the disclosure typically are sterileand stable under conditions of manufacture, storage and use. Sterilesolutions can be prepared by incorporating the compound in the requiredamount in an appropriate solvent with one or a combination ofingredients enumerated herein, as required, followed by filteredsterilization. Generally, dispersions are prepared by incorporating thecompound and/or other biologically active agent into a sterile vehiclethat contains a basic dispersion medium and the required otheringredients from those enumerated herein. In the case of sterilepowders, methods of preparation include vacuum drying and freeze-dryingwhich yields a powder of the compound plus any additional desiredingredient from a previously sterile-filtered solution thereof. Theprevention of the action of microorganisms can be accomplished byvarious antibacterial and antifungal agents, for example, parabens,chlorobutanol, phenol, sorbic acid, thimerosal, and the like.

In accordance with the various treatment methods of the disclosure, thecalcium channel agonist can be delivered to a subject in a mannerconsistent with conventional methodologies associated with management ofthe disorder for which treatment or prevention is sought. In accordancewith the disclosure herein, a prophylactically or therapeuticallyeffective amount of the calcium channel agonist is administered to asubject in need of such treatment for a time and under conditionssufficient to inhibit, and/or ameliorate a selected disease or conditionor one or more symptom(s) thereof.

The administration of the calcium channel agonist of the disclosure canbe for either prophylactic or therapeutic purpose. When providedprophylactically, the calcium channel agonist is provided in advance ofany symptom. The prophylactic administration of the calcium channelagonist serves to inhibit or ameliorate any subsequent disease process.When provided therapeutically, the calcium channel agonist is providedat (or shortly after) the onset of a symptom of disease.

For prophylactic and therapeutic purposes, the calcium channel agonistcan be administered to the subject by the oral route or in a singlebolus delivery, via continuous delivery (for example, continuoustransdermal, mucosal or intravenous delivery) over an extended timeperiod, or in a repeated administration protocol (for example, by anhourly, daily or weekly, repeated administration protocol). Thetherapeutically effective dosage of the calcium channel agonist can beprovided as repeated doses within a prolonged prophylaxis or treatmentregimen that will yield clinically significant results to alleviate oneor more symptoms or detectable conditions associated with a targeteddisease or condition as set forth herein. Determination of effectivedosages in this context is typically based on animal model studiesfollowed up by human clinical trials and is guided by administrationprotocols that significantly reduce the occurrence or severity oftargeted disease symptoms or conditions in the subject. Suitable modelsin this regard include, for example, murine, rat, avian, porcine,feline, non-human primate, and other accepted animal model subjectsknown in the art. Alternatively, effective dosages can be determinedusing in vitro models. Using such models, only ordinary calculations andadjustments are required to determine an appropriate concentration anddose to administer a therapeutically effective amount of the calciumchannel agonist (for example, amounts that are effective to elicit adesired increase in calcium channel activity or alleviate one or moresymptoms of a targeted disease).

The actual dosage of the calcium channel agonist will vary according tofactors such as the disease indication and particular status of thesubject (for example, the subject's age, size, fitness, extent ofsymptoms, susceptibility factors, and the like), time and route ofadministration, other drugs or treatments being administeredconcurrently, as well as the specific pharmacology of the compound foreliciting the desired activity or biological response in the subject.Dosage regimens can be adjusted to provide an optimum therapeuticresponse. A therapeutically effective amount is also one in which anytoxic or detrimental side effects of the compound and/or otherbiologically active agent is outweighed in clinical terms bytherapeutically beneficial effects. A non-limiting range for atherapeutically effective amount of a compound and/or other biologicallyactive agent within the methods and formulations of the disclosure isabout 0.01 mg/kg body weight to about 20 mg/kg body weight, such asabout 0.05 mg/kg to about 5 mg/kg body weight, or about 0.2 mg/kg toabout 2 mg/kg body weight.

Dosage can be varied by the attending clinician to maintain a desiredconcentration at a target site (for example, the lungs or systemiccirculation). Higher or lower concentrations can be selected based onthe mode of delivery, for example, trans-epidermal, rectal, oral,pulmonary, or intranasal delivery versus intravenous or subcutaneousdelivery. Dosage can also be adjusted based on the release rate of theadministered formulation, for example, of an intrapulmonary spray versuspowder, sustained release oral versus injected particulate ortransdermal delivery formulations, and so forth.

The calcium channel agonists disclosed herein may be co-administeredwith an additional therapeutic agent. Such agents include, but are notlimited to, acetylcholinesterase inhibitors, immunosuppressants,intravenous immunoglobulins, glucocorticoids, ascorbic acid, anti-canceragents (e.g., if the calcium channel dysfunction is caused by underlyingmalignancy), potassium channel blockers, copper supplementation (if thecalcium channel dysfunction is caused by copper deficiency), analgesics,antidepressants, muscle relaxants, and combinations thereof. The calciumchannel agonist also may be co-administered with adjunct therapies, suchas plasmapheresis, ventilator assistance (e.g., for Guillain-Barrésyndrome or amyotrophic lateral sclerosis), braces, physical therapy,occupational therapy, or combinations thereof. Co-administration may beperformed simultaneous or sequentially.

In certain embodiments, when the condition to be treated is LEMS, thecalcium channel agonist may be co-administered with a therapeuticallyeffective amount of 3,4-diaminopyridine (DAP, a potassium channelblocker). In combination, a calcium channel agonist according to generalformula I and DAP may exert synergistic effects on transmitter releasewhen both are applied at concentrations that are lower than what isrequired for effects when given alone, thereby lowering thetherapeutically effective amount of one or both compounds. Because DAPbroadens the presynaptic action potential, calcium channels will openfor a longer period of time during the action potential, and the openchannel binding of the calcium channel agonist would be expected tooccur more frequently. Therefore, DAP administered in combination withan embodiment of the disclosed calcium channel agonists may result in astronger effect that provides an even greater reversal of neuromuscularweakness.

The instant disclosure also includes kits, packages and multi-containerunits containing the herein described pharmaceutical compositions,active ingredients, and/or means for administering the same for use inthe prevention and treatment of diseases and other conditions inmammalian subjects. The calcium channel agonist may be formulated in apharmaceutical preparation for delivery to a subject. The calciumchannel agonist is optionally contained in a bulk dispensing containeror unit or multi-unit dosage form. Optional dispensing means can beprovided, for example a pulmonary or intranasal spray applicator.Packaging materials optionally include a label or instruction indicatingfor what treatment purposes and/or in what manner the pharmaceuticalagent packaged therewith can be used.

B. An Experimental Tool for Studying Ca²⁺ channel Subtypes

Historically, calcium channel gating modifiers have been valuableexperimental tools. Because voltage-gated calcium channels have arelatively small conductance and brief mean open time, single channelgating has been relatively difficult to study in detail. Further,studies of physiological effects of calcium channels can be aided by theuse of gating modifiers. Relatively few calcium channel agonists havebeen identified (the most well-known among them is BayK 8644), and thosethat have been developed only bind selectively to L-type calciumchannels. These L-type agonists have provided an experimentalopportunity to increase mean open time for L-type calcium channels. Withthis tool, investigators have been better able to determine conductancein physiological calcium concentrations, test for a role of L-typechannels in the regulation of transmitter release at synapses, study thestructural motifs within the L-type calcium channel that regulategating, study the influence of ion permeation on gating, and examinecell signaling that employs L-type calcium channels, among many otherexperimental uses.

Independent of its therapeutic potential for treatment of diseasescharacterized by neuromuscular weakness, a selective and potent Ca²⁺channel agonist of the P/Q- and N-type Ca²⁺ channels would serve as animportant experimental tool for studying the basic properties of theseCa²⁺ channel subtypes. Just as the L-type Ca²⁺ channel agonists BayK8644 and FPL64176 were important in studies of L-type channel gating,conductance, and kinetics, an agonist of the P/Q- and N-type channelswould be equally as useful in the study of their properties.Furthermore, embodiments of the disclosed compounds may serve as auseful probe molecule in studies of the calcium control of chemicaltransmitter release. Even though (R)-roscovitine is an agonist of theP/Q- and N-type channel subtypes, certain embodiments of the disclosedcalcium channel agonists (e.g., compound 13x) are more selective andpotent than (R)-roscovitine, and thus likely to be more useful forstudies on basic P/Q- and N-type Ca²⁺ channel function.

V. Examples

General Information:

All moisture and air-sensitive reactions were performed usingsyringe-septum cap techniques under an inert atmosphere (N₂ or Ar) inglassware that was dried in an oven at 140° C. for at least 2 h prior touse. Reactions carried out at a temperature below 0° C. employed aCO₂/acetone bath. All reagents and solvents were used as received unlessotherwise specified. Triethylamine, N,N-dimethylaniline and pyridinewere distilled over CaH₂. THF and Et₂O were distilled oversodium/benzophenone ketyl. Dichloromethane and toluene were purifiedusing an alumina column filtration system. Anhydrous MeOH and Et₂O werepurchased from Acros Organics and Fisher Scientific, respectively.Anhydrous DMF was purchased from Acros Organics or distilled and storedover 4 A molecular sieves. Analytical thin-layer chromatography (TLC)was performed on pre-coated SiO₂ 60 F254 plates (250 μm layer thickness)available from Merck. Visualization was accomplished by UV irradiationat 254 nm and/or by staining with Vaughn's reagent (4.8 g(NH₄)₆Mo₇O₂₄.4H₂O and 0.2 g Ce(SO₄)₂.4H₂O in 100 mL of a 3.5 N H₂SO₄solution), a KMnO₄ solution (1.5 g KMnO₄ and 1.5 g K₂CO₃ in 100 mL of a0.1% NaOH solution), a ninhydrin solution (2 g ninhydrin in 100 mL ofethanol (EtOH)), a PMA solution (5 g of phosphomolybdic acid in 100 mLof EtOH), or a p-anisaldehyde solution (2.5 mL of p-anisaldehyde, 2 mLof acetic acid and 3.5 mL of conc. aq. H₂SO₄ in 100 mL of EtOH).Preparative thin-layer chromatography was performed on pre-coated SiO₂GF (UV₂₅₄) 1000 microns (20×20 cm) plates available from Analtech. Flashcolumn chromatography was performed using SiO₂ 60 (particle size0.040-0.055 mm, 230-400 mesh, or Silicycle SiliaFlash® P60, 40-63 μm).Melting points were determined on a Meltemp capillary melting pointapparatus fitted with a Fluke 51 II digital thermometer. Infraredspectra were recorded on a Smiths IdentifyIR ATR spectrometer or aPerkin Elmer Spectrum 100 FT-IR spectrometer using the Universal ATRSampling Accessory for both oil and solid compounds. ¹H NMR and ¹³C NMRspectra were obtained on Bruker Avance 300, 400 or 600 instruments at300/75 MHz, 400/100 MHz or 600/150 MHz, respectively. Chemical shiftswere reported in parts per million (ppm) as referenced to residualsolvent. ¹H NMR spectra were tabulated as follows: chemical shift,multiplicity (app=apparent, b=broad, s=singlet, d=doublet, t=triplet,q=quartet, quint=quintuplet, sext=sextuplet, sept=septuplet,m=multiplet), number of protons, coupling constant(s). ¹³C NMR spectrawere obtained using a proton-decoupled pulse sequence. Mass spectra wereobtained on a Waters Autospec double focusing mass spectrometer (EI) ora Waters Q-Tof mass spectrometer (ESI).

Compound Synthesis and Characterization:

2,6-Dichloro-9-propyl-9H-purine (15a). To a solution of2,6-dichloro-9H-purine 14 (0.490 mg, 2.59 mmol) in anhydrous DMSO (3.0mL) was added K₂CO₃ (1.10 g, 7.96 mmol) followed by 1-bromopropane (1.62g, 13.1 mmol) at 16-18° C. (isopropanol (i-PrOH) bath in a Dewar flaskcovered with aluminum foil). The reaction mixture was stirred at 16-18°C. for 17 h, quenched with water and extracted with ethyl acetate(EtOAc). The combined organic layers were washed with brine, dried(MgSO4), concentrated, and purified by chromatography on SiO2 (hexanes,100%, to hexanes/EtOAc, 1:1) to yield 15a (0.465 g, 2.01 mmol, 78%yield) as an off-white solid: IR (ATR, neat) 3677, 3078, 2974, 2939,2880, 1596, 1553, 1496, 1466, 1442, 1408, 1383, 1370, 1347, 1312, 1270,1229, 1196, 1180, 1141, 1084, 957, 901, 875, 860, 812, 785, 774, 681cm⁻¹; ¹H NMR (600 MHz, CDCl₃) δ 8.10 (s, 1 H), 4.21 (t, 2 H, J=7.2 Hz),1.93 (sext, 2 H, J=7.4 Hz), 0.94 (t, 3 H, J=7.5 Hz); ¹³C NMR (151 MHz,CDCl₃) δ 153.2, 152.7, 151.5, 146.0, 46.2, 23.1, 11.1.

N-(Biphenyl-4-ylmethyl)-2-chloro-9-propyl-9H-purin-6-amine (16a). To asolution of 15a (58.0 mg, 0.251 mmol) in n-butanol (n-BuOH) (1.0 mL)were added 4-phenylbenzylamine (52.0 mg, 0.267 mmol) and triethylamine(40.6 mg, 0.402 mmol) under an N₂ atmosphere at room temperature. Thereaction mixture was heated in a microwave at 120° C. for 20 min. Thesolvent was evaporated, and the crude residue was dissolved in EtOAc andwashed with water. The aqueous phase was further extracted with EtOAc,and the combined organic extracts were dried (MgSO₄) and concentrated toyield a colorless solid, which was resuspended (hexanes/Et₂O, 3:1),filtered, triturated (hexanes/Et₂O, 3:1) and dried under high-vacuum toyield 16a (86.2 mg, 0.228 mmol, 91% yield) as a colorless solid: IR(ATR, neat) 3145, 2964, 1619, 1577, 1304, 1254 cm⁻¹; ¹H NMR (600 MHz,CDCl₃) δ 7.56 (d, 2 H, J=8.4 Hz), 7.56 (d, 2 H, J=7.8 Hz), 7.47-7.40 (m,5 H), 7.35 (t, 1 H, J=7.5 Hz), 6.88 (bm, 1 H), 4.87 (bs, 2 H), 4.05 (t,2 H, J=6.6 Hz), 1.85 (sext, 2 H, J=7.4 Hz), 0.91 (t, 3 H, J=6.9 Hz); ¹³CNMR (151 MHz, CDCl₃) δ 155.3, 154.7, 150.4, 140.7, 140.4, 137.2, 128.9,128.6, 127.5 (2 C), 127.2, 118.8, 45.6, 44.5, 23.4, 11.2.

(R)-2-(6-(Biphenyl-4-ylmethylamino)-9-propyl-9H-purin-2-ylamino)butan-1-ol(13a). A mixture of 16a (50.0 mg, 0.128 mmol) and(R)-(−)-2-amino-1-butanol (60.9 mg, 0.642 mmol) was heated in amicrowave vial immersed in an oil bath at 170° C. for 8 h. The reactionmixture was cooled to room temperature, diluted with water, andextracted with EtOAc (4×). The combined organic layers were washed withwarmed water (50-55° C., 2×), dried (MgSO4), concentrated, and driedunder high vacuum at 50° C. (oil bath) for 2 h to yield a yellow solid.Upon the addition of Et₂O to the yellow solid, an off-white solidprecipitated. The solid was further washed with Et₂O (3×) and driedunder high-vacuum at 40° C. overnight to yield 13a (32.6 mg, 0.0757mmol, 59%) as an off-white solid: Mp 130-131° C.; IR (ATR, neat) 3270,2962, 2931, 1599, 1488, 1349 cm⁻¹; ¹H NMR (600 MHz, CDCl₃) δ 7.57 (appd, 2 H, J=7.8 Hz), 7.54 (app d, 2 H, J=7.8 Hz), 7.47-7.38 (m, 4 H),7.37-7.28 (m, 2 H), 6.46 (bs, 1 H), 5.26-5.08 (bm, 1 H), 4.97 (s, 1 H),4.91-4.66 (bm, 2 H), 3.92 (t, 2 H, J=6.9 Hz), 3.90-3.85 (bm, 1 H), 3.81(bd, 1 H, J=12.0 Hz), 3.63 (app t, 1 H, J=9.0 Hz), 1.82 (sext, 2 H,J=7.2 Hz), 1.67-1.49 (m, 2 H), 1.00 (t, 3 H, J=7.2 Hz), 0.91 (t, 3 H,J=7.2 Hz); ¹³C NMR (151 MHz, CDCl₃) δ 160.2, 154.9, 150.7, 140.7, 140.2,137.9, 137.1, 128.7, 128.1, 127.2, 127.0, 114.3, 68.2, 56.2, 45.0, 44.0,25.0, 23.1, 11.2, 10.9; HRMS (ES⁺) m/z calcd for C₂₅H₃₁N₆O [M+H]⁺431.2559, found 431.2532.

2-Chloro-N-(2,2-diphenylethyl)-9-propyl-9H-purin-6-amine (16b). To asolution of 15a (71.0 mg, 0.307 mmol) in n-BuOH (1.0 mL) was addedaminodiphenylmethane (61.5 mg, 0.326 mmol) and triethylamine (50.1 mg,0.495 mmol) under an N₂ atmosphere. The reaction mixture was heated in amicrowave reactor at 120° C. for 20 min. The solvent was evaporated, andthe residue was dissolved in EtOAc and washed with water. The aqueousphase was further extracted with EtOAc, and the combined organicextracts were dried (MgSO₄) and concentrated to yield a colorless solid,which was resuspended (hexanes/Et₂O, 3:1), filtered, and triturated(hexanes/Et₂O, 3:1) to obtain an off-white solid. The filtrate wasconcentrated, resuspended (hexanes/Et₂O, 3:1), and filtered to obtainadditional product. After drying on high-vacuum, 16b (74.4 mg, 0.197mmol, 65%) was obtained as an off-white solid: IR (ATR, neat) 3250,2964, 1612, 1574, 1304, 1218 cm⁻¹; ¹H NMR (600 MHz, CDCl₃) δ 7.54 (bs, 1H), 7.35-7.20 (m, 9 H), 7.17 (s, 1 H), 6.78 (d, 1 H, J=6.6 Hz),4.09-3.90 (bm, 2 H), 1.95-1.69 (bm, 2 H), 0.90 (t, 3 H, J=6.9 Hz); ¹³CNMR (151 MHz, CDCl₃) δ 154.4, 154.2, 150.3, 141.3, 140.4, 127.7, 127.5,127.3, 118.5, 57.1, 45.4, 23.2, 11.0; LCMS (ES⁺) m/z calcd forC₂₁H₂₁N₅Cl [M+H]⁺ 378.1, found 378.1.

(R)-2-(6-(2,2-Diphenylethylamino)-9-propyl-9H-purin-2-ylamino)butan-1-ol(13b). A mixture of 16b (50.0 mg, 0.128 mmol) and(R)-(−)-2-amino-1-butanol (60.9 mg, 0.642 mmol) were heated in a vialimmersed in an oil bath at 170° C. for 8 h. The reaction mixture wascooled to room temperature, diluted with water, and extracted with EtOAc(4×). The combined organic phases were washed with warm water (50-55°C., 2×), dried (MgSO₄), concentrated, and dried under high-vacuum at 50°C. (oil bath) for 2 h to yield a solid-yellow oil. Upon addition ofEt₂O, an off-white solid precipitated. The solid was rinsed with Et₂O(3×) and dried under high-vacuum at 40° C. to yield 13b (36.9 mg, 0.0857mmol, 67%) as an off-white solid: Mp 160-163° C.; IR (ATR, neat) 3269,2960, 1606, 1556, 1439 cm⁻¹; ¹H NMR (600 MHz, CDCl₃) δ 7.40-7.26 (m, 8H), 7.25-7.16 (m, 2 H), 6.64-6.29 (m, 2 H), 4.80 (d, 1 H, J=4.8 Hz),3.89 (t, 2 H, J=6.9 Hz), 3.79-3.69 (bm, 1 H), 3.69-3.61 (bm, 1 H),3.55-3.41 (bm, 1 H), 1.79 (sext, 2 H, J=6.6 Hz), 1.61-1.37 (m, 2 H),1.05-0.90 (m, 6 H); ¹³C NMR (151 MHz, CDCl₃) δ 160.0, 153.9, 142.2,137.2, 128.5 (2 C), 127.6, 127.2 (2 C), 114.3, 67.7, 57.9, 56.0, 45.0,24.8, 23.1, 11.2, 10.8; HRMS (ES⁺) m/z calcd for C₂₅H₃₁N₆O [M+H]⁺431.2559, found 431.2596.

2-Chloro-N-phenethyl-9-propyl-9H-purin-6-amine (16c). To a solution of15a (58.0 mg, 0.251 mmol) in n-BuOH (1.0 mL) was added phenethylamine(32.8 mg, 0.269 mmol) and triethylamine (40.6 mg, 0.402 mmol) under anN₂ atmosphere. The reaction mixture was heated under microwaveirradiation at 120° C. for 30 min. n-BuOH was evaporated and the residuewas dissolved in EtOAc and washed with water. The aqueous phase wasfurther extracted with EtOAc and the combined organic layers were dried(MgSO₄) and concentrated to yield a colorless solid, which wasresuspended (hexanes/Et₂O, 3:1, filtered, triturated (hexanes/Et₂O, 3:1)and dried under high-vacuum to yield 16c (67.0 mg, 0.212 mmol, 85%) asan amorphous off-white solid: IR (ATR, neat) 3218, 2960, 1620, 1576,1355, 1307, 1232 cm⁻¹; ¹H NMR (600 MHz, CDCl₃) δ 7.47-7.30 (m, 1 H),7.25-7.19 (m, 2 H), 7.19-7.09 (m, 3 H), 6.91-6.63 (m, 1 H), 4.15-3.98(bm, 2 H), 3.953.67 (bm, 2 H), 3.00-2.87 (bm, 2 H), 1.98-1.78 (bm, 2 H),0.92 (bt, 3 H, J=6.3 Hz); ¹³C NMR (151 MHz, CDCl₃) δ 155.1, 154.4,149.9, 139.8, 138.8, 128.7, 128.4, 126.2, 118.5, 45.3, 41.8, 35.4, 23.2,11.0.

(R)-2-(6-(Phenethylamino)-9-propyl-9H-purin-2-ylamino)butan-1-ol (13c).A mixture of 16c (50.0 mg, 0.158 mmol) and (R)-(−)-2-amino-1-butanol(76.0 mg, 0.801 mmol) was heated in a microwave vial immersed in an oilbath at 170° C. for 7 h. The reaction mixture was cooled to roomtemperature, diluted with water, and extracted with EtOAc (3×). Thecombined organic phases were washed with warm water (50-55° C., 2×),dried (MgSO₄), concentrated, and dried under high-vacuum at 50° C. (oilbath) for 2 h to yield an oily yellow residue. Upon the addition of Et₂Oand a few drops of hexanes, an off-colorless solid precipitated. Thesolid was rinsed (Et₂O, 3×) by pipetting out the supernatant, and thesolid was dried under high-vacuum at 40° C. overnight to yield 13c (18.7mg, 0.0508 mmol, 32%) as an off-white solid: Mp 105-107° C.; IR (ATR,neat) 3276, 2956, 2929, 1603, 1520 cm⁻¹; ¹H NMR (600 MHz, CDCl₃) δ 7.43(bs, 1 H), 7.407.32 (m, 2 H), 7.32-7.22 (m, 3 H), 5.80 (bs, 1 H), 5.40(bs, 1 H), 5.04-4.84 (bm, 1 H), 3.99 (t, 2 H, J=6.6 Hz), 3.97-3.91 (bm,1 H), 3.91-3.75 (bm, 3 H), 3.69 (t, 1 H, J=9.0 Hz), 2.99 (t, 2 H, J=6.3Hz), 1.89 (sext, 2 H, J=6.6 Hz), 1.75-1.55 (m, 2 H), 1.08 (t, 3 H, J=7.2Hz), 0.98 (t, 3 H, J=7.2 Hz); ¹³C NMR (151 MHz, CDCl₃) δ 160.2, 154.9,139.0, 137.0, 128.8, 128.6, 126.4, 114.4, 68.6, 56.4, 45.0, 41.7, 35.9,25.0, 23.2, 11.2, 10.9; HRMS (ES⁺) m/z calcd for C₂₀H₂₉N₆O [M+H]⁺369.2403, found 369.2422.

N-Benzyl-2-chloro-9-propyl-9H-purin-6-amine (16d). To a solution of 15a(63.0 mg, 0.273 mmol) in n-BuOH (1.0 mL) was added benzylamine (31.4 mg,0.287 mmol) and triethylamine (43.6 mg, 0.430 mmol) under an N₂atmosphere. The reaction mixture was heated under microwave irradiationat 120° C. for 30 min. n-BuOH was evaporated, and the residue wasdissolved in EtOAc and washed with water. The aqueous phase was furtherextracted with EtOAc and the combined organic extracts were dried(MgSO₄) and concentrated to yield a colorless solid, which wasresuspended (hexanes/Et₂O, 3:1), filtered, triturated (hexanes/Et₂O,3:1), and dried under high-vacuum to yield 16d (66.3 mg, 0.220 mmol,81%) as a colorless solid: IR (ATR, neat) 3189, 2966, 1623, 1304, 1253cm⁻¹; ¹H NMR (600 MHz, CDCl₃) δ 7.44-7.27 (m, 6 H), 7.10-6.75 (bm, 1 H),4.82 (bs, 2 H), 4.20-4.00 (bm, 2 H), 1.95-1.80 (bm, 2 H), 0.93 (t, 3 H,J=6.6 Hz); ¹³C NMR (151 MHz, CDCl₃) δ 155.1, 154.5, 150.2, 140.2, 138.0,128.6, 127.9, 127.5, 118.6, 45.4, 44.6, 23.3, 11.1.

(R)-2-(6-(Benzylamino)-9-propyl-9H-purin-2-ylamino)butan-1-ol (13d). Amixture of 16d (50.0 mg, 0.166 mmol) and (R)-(−)-2-amino-1-butanol (78.9mg, 0.831 mmol) was heated in a microwave vial immersed in an oil bathat 170° C. for 7 h. The reaction mixture was cooled to room temperature,diluted with water, and extracted with EtOAc (3×). The combined organicphases were washed with warm water (50-55° C., 2×) dried (MgSO₄),concentrated, and dried under high-vacuum at 50° C. (oil bath) for 2 hto yield a yellow solid. Upon the addition of Et₂O to the yellow solid,an off-white solid precipitated. The solid was rinsed (Et₂O, 3×) bypipetting out the supernatant and dried under high-vacuum at 40° C.overnight to yield 13d (38.6 mg, 0.109 mmol, 66%) as an off-white solid:Mp 153-155° C.; IR (ATR, neat) 3262, 3201, 2961, 1624, 1603, 1513 cm⁻¹;¹H NMR (CDCl₃, 600 MHz) δ 7.23-7.40 (m, 6 H), 6.22 (bs, 1 H), 5.19 (bs,1 H), 4.88-4.96 (m, 1 H), 4.75 (bs, 2 H), 3.92-3.97 (m, 2 H), 3.85-3.92(m, 1 H), 3.81 (d, 1 H, J=10.8 Hz), 3.62 (t, 1 H, J=8.7 Hz), 1.90-1.80(m, 2 H), 1.70-1.50 (m, 2 H), 1.02 (t, 3 H, J=7.2 Hz), 0.94 (t, 3 H,J=6.6 Hz); ¹³C NMR (CDCl₃, 150 MHz) δ 160.2, 154.8, 150.6, 138.7, 137.1,128.5, 127.7, 127.3, 114.3, 68.4, 56.3, 45.0, 44.4, 25.0, 23.2, 11.2,10.9; HRMS (ES) m/z calcd for C₁₉H₂₆N₆O [M+H]355.2246, found 355.2241.

2-Chloro-N-phenyl-9-propyl-9H-purin-6-amine (16e). To a solution of 15a(72.0 mg, 0.312 mmol) in n-BuOH (1.0 mL) were added aniline (30.2 mg,0.324 mmol) and triethylamine (50.1 mg, 0.495 mmol) under an N₂atmosphere. The reaction mixture was heated under microwave irradiationat 120° C. for 30 min. n-BuOH was evaporated, and the residue wasdissolved in EtOAc and washed with water. The aqueous phase was furtherextracted with EtOAc, and the combined organic extracts were dried(MgSO₄) and concentrated to yield a colorless solid. The solid wasresuspended (hexanes/Et₂O, 3:1), filtered, triturated (hexanes/Et₂O,3:1), and dried under high-vacuum to yield 16e (52.7 mg, 0.183 mmol,59%) as an off-white amorphous solid: IR (ATR, neat) 3179, 2967, 1611,1572, 1346, 1301 cm⁻¹; ¹H NMR (CDCl₃, 600 MHz) δ 8.31 (bs, 1 H),7.677.81 (m, 3 H), 7.36 (t, 2 H, J=7.8 Hz), 7.12 (t, 1 H, J=7.2 Hz),4.11 (t, 2 H, J=7.2 Hz), 1.89 (sext, 2 H, J=7.2 Hz), 0.94 (t, 3 H, J=7.2Hz); ¹³C NMR (CDCl₃, 150 MHz) δ 153.9, 152.3, 150.7, 141.0, 138.0,128.9, 123.9, 120.3, 119.1, 45.5, 23.2, 11.0.

(R)-2-(6-(Phenylamino)-9-propyl-9H-purin-2-ylamino)butan-1-ol (13e). Amixture of 16e (41.0 mg, 0.142 mmol) and (R)-(−)-2-amino-1-butanol (68.4mg, 0.721 mmol) was heated in a microwave vial immersed in an oil bathat 170° C. for 12 h. The reaction mixture was cooled to roomtemperature, diluted with water, and extracted with EtOAc (3×). Thecombined organic phases were washed with warm water (50-55° C., 2×),dried (MgSO₄), concentrated, and dried under high-vacuum to yield alight-green solid. The crude solid was adsorbed onto SiO₂ and purifiedby chromatography on SiO₂ (hexanes/EtOAc, 1:1 to EtOAc, 100% with 1%Et₃N to 10% MeOH in EtOAc with 1% Et₃N) to yield 13e (23.6 mg, 0.0693mmol, 49%) as an off-white solid: Mp 190-194° C.; IR (ATR, neat) 3338,2970, 1644, 1583, 1498, 1474, 1442 cm⁻¹′; ¹H NMR (CDCl₃, 600 MHz) δ 7.75(d, 2 H, J=7.8 Hz), 7.65 (bs, 1 H), 7.51 (s, 1 H), 7.35 (t, 2 H, J=7.8Hz), 7.08 (t, 1 H, J=7.8 Hz), 5.03 (d, 1 H, J=6.6 Hz), 3.93-4.02 (m, 3H), 3.87 (dd, 1 H, J=10.8, 1.8 Hz), 3.68 (dd, 1 H, J=10.8, 7.2 Hz), 1.87(sext, 2 H, J=7.2 Hz), 1.57-1.73 (m, 2 H), 1.05 (t, 3 H, J=7.2 Hz), 0.96(t, 3 H, J=7.2 Hz); ¹³C NMR δ (CDCl₃, 150 MHz) δ 159.9, 152.4, 150.9,139.0, 137.6, 128.9, 123.0, 120.0, 114.8, 56.1, 45.1, 29.7, 25.0, 23.2,11.2, 10.9; HRMS (EI) m/z calcd for C₁₈H₂₄N₆O 340.2012, found 340.2009.

2-Chloro-9-propyl-N-(pyridin-3-ylmethyl)-9H-purin-6-amine (16f). To asolution of 15a (58.0 mg, 0.251 mmol) in n-BuOH (1.0 mL) were added3-(aminomethyl)-pyridine (28.6 mg, 0.265 mmol) and triethylamine (40.6mg, 0.402 mmol) under an N₂ atmosphere. The reaction mixture was heatedunder microwave irradiation at 120° C. for 30 min. n-BuOH wasevaporated, and the residue was dissolved in EtOAc and washed withwater. The aqueous phase was further extracted with EtOAc, and thecombined organic extracts were dried (MgSO₄) and concentrated to yield acolorless solid. The solid was resuspended (hexanes/Et₂O, 3:1),filtered, triturated (hexanes/Et₂O, 3:1), and dried under high-vacuum toyield 16f (53.9 mg, 0.178 mmol, 71%) as a yellow amorphous solid: IR(ATR, neat) 3155, 2964, 1626, 1572, 1308, 1232 cm⁻¹; ¹H NMR (CDCl₃, 600MHz) δ 8.57 (s, 1 H), 8.52-8.44 (m, 1 H), 7.64 (d, 1 H, J=7.8 Hz), 7.50(bs, 1 H), 7.33 (s, 1 H), 7.18 (dd, 1 H, J=7.2, 4.8 Hz), 4.79 (bs, 2 H),4.05 (t, 2 H, J=7.2 Hz), 1.83 (sext, 2 H, J=7.2 Hz), 0.89 (t, 3 H, J=7.2Hz); ¹³C NMR (CDCl₃, 150 MHz) δ 154.9, 154.3, 151.0, 149.2, 148.8,140.2, 135.5, 133.7, 123.4, 118.5, 45.4, 41.8, 23.2, 11.0.

(R)-2-(9-Propyl-6-(pyridin-3-ylmethylamino)-9H-purin-2-ylamino)butan-1-ol(13f). A mixture of 16f (35.0 mg, 0.116 mmol) and(R)-(−)-2-amino-1-butanol (55.1 mg, 0.581 mmol) was heated in amicrowave vial immersed in an oil bath at 170° C. for 12 h. The reactionmixture was cooled to room temperature, diluted with water, andextracted with EtOAc (3×). The combined organic phases were washed withwarm water (50-55° C., 2×), dried (MgSO₄), concentrated, and dried underhigh-vacuum to yield an oily yellow residue. Upon the addition of Et₂Oand hexanes (drops), a sticky, yellow solid precipitated. The solid wascarefully crushed with a glass rod to yield an off-white solid, whichwas rinsed with Et₂O (3×) by pipetting out the supernatant and driedunder high-vacuum overnight to yield 13f (27.0 mg, 0.0760 mmol, 66%) asan off-white solid: Mp 129-132° C.; IR (ATR, neat) 3257, 3209, 2964,1601, 1530, 1477 cm⁻¹; ¹H NMR (CDCl₃, 600 MHz) δ 8.68 (s, 1 H), 8.54 (d,1 H, J=4.8 Hz), 7.73 (d, 1 H, J=7.8 Hz), 7.46 (s, 1 H), 7.30-7.28 (m, 1H), 5.99 (bs, 1 H), 4.89-4.86 (m, 1 H), 4.81 (bs, 2 H), 4.00 (t, 2 H,J=7.2 Hz), 3.92-3.86 (m, 1 H), 3.81 (d, 1 H, J=10.2 Hz), 3.63 (dd, 1 H,J=10.2, 7.8 Hz), 1.89 (sext, 2 H, J=7.2 Hz), 1.73-1.63 (m, 2 H),1.63-1.53 (m, 1 H), 1.04 (t, 3 H, J=7.2 Hz), 0.97 (t, 3 H, J=7.2 Hz);¹³C NMR (CDCl₃, 150 MHz) δ 160.1, 154.7, 150.9, 149.3, 148.6, 137.3,135.3, 134.5, 123.4, 114.3, 67.9, 56.1, 45.1, 41.8, 24.9, 23.1, 11.2,10.9; HRMS (EI) m/z calcd for C₁₈H₂₅N₇O 355.2121, found 355.2124.

2-Chloro-N-(cyclopropylmethyl)-9-propyl-9H-purin-6-amine (16g). To asolution of 15a (80.0 mg, 0.346 mmol) in n-BuOH (1 mL) were addedaminomethyl-cyclopropane (28.7 mg, 0.391 mmol) and triethylamine (56.6mg, 0.560 mmol) under an N₂ atmosphere. The reaction mixture was heatedunder microwave irradiation at 120° C. for 30 min. n-BuOH wasevaporated, and the residue was dissolved in EtOAc and washed withwater. The aqueous phase was further extracted with EtOAc, and thecombined organic extracts were dried (MgSO₄), filtered, concentrated,and dried under high-vacuum overnight to yield 16g (87.1 mg, 0.328 mmol,95%) as an amorphous colorless solid: IR (ATR, neat) 3260, 2970, 1621,1578, 1302, 1253 cm⁻¹; ¹H NMR (CDCl₃, 600 MHz) δ 7.67 (s, 1 H), 6.26(bs, 1 H), 4.06 (t, 2 H, J=7.2 Hz), 3.45-3.35 (m, 2 H), 1.84 (sext, 2 H,J=7.2 Hz), 1.10-1.00 (m, 1 H), 0.89 (t, 2 H, J=7.2 Hz), 0.48 (dd, 2 H,J=13.8, 4.8 Hz), 0.23 (dd, 2 H, J=9.6, 4.8 Hz); ¹³C NMR (CDCl₃, 150 MHz)δ 155.0, 154.3, 149.9, 139.8, 118.4, 60.2, 45.6, 45.3, 23.2, 10.9, 10.5,3.4.

(R)-2-(6-(Cyclopropylmethylamino)-9-propyl-9H-purin-2-ylamino)butan-1-ol(13g). A mixture of 15a (50.0 mg, 0.188 mmol) and(R)-(−)-2-amino-1-butanol (89.2 mg, 0.941 mmol) was heated in a pressuretube immersed in an oil bath at 170° C. for 10 h. The reaction mixturewas cooled to room temperature, diluted with water, and extracted withEtOAc (3×). The combined organic phases were washed with warm water(50-55° C., 2×), dried (MgSO₄), concentrated, and dried underhigh-vacuum to yield a yellow solid. Upon the addition of Et₂O to thesolid, an off-white solid was precipitated. The solid was rinsed (Et₂O,3×) by pipetting out the supernatant and dried under high-vacuum toyield 13 g (37.4 mg, 0.117 mmol, 62%) as an off-white solid: Mp 146-149°C.; IR (ATR, neat) 3319, 2966, 2847, 1611, 1489 cm⁻¹; ¹H NMR (CDCl₃, 600MHz) δ 7.42 (s, 1 H), 5.81 (bs, 1 H), 5.51 (bs, 1 H), 4.96-4.88 (m, 1H), 3.94 (t, 2 H, J=7.2 Hz), 3.91-3.85 (m, 1 H), 3.82 (d, 1 H, J=10.2Hz), 3.63 (dd, 1 H, J=10.2, 8.4 Hz), 3.38 (bs, 2 H), 1.84 (sext, 2 H,J=7.2 Hz), 1.68-1.49 (m, 2 H), 1.15-1.05 (m, 1 H), 1.01 (t, 3 H, J=7.2Hz), 0.92 (t, 3 H, J=7.2 Hz), 0.55-0.50 (m, 2 H), 0.28-0.23 (m, 2 H);¹³C NMR (CDCl₃, 150 MHz) δ 160.2, 154.8, 150.5, 136.9, 114.2, 68.4,56.3, 45.4, 45.0, 25.0, 23.1, 11.1, 10.9, 10.8, 3.4; HRMS (EI) m/z calcdfor C₁₆H₂₆N₆O 318.2168, found 318.2168.

2,6-Dichloro-9-methyl-9H-purine (15b). To a solution of2,6-dichloro-9H-purine (0.120 g, 0.635 mmol) in anhydrous DMF (1.0 mL)was added K₂CO₃ (0.270 g, 1.95 mmol) followed by iodomethane (0.20 mL,3.21 mmol) at 0° C. The reaction mixture was stirred for 5 h at 0° C.,quenched with water and extracted with EtOAc. The organic layers werecombined, washed with brine, dried (MgSO4), concentrated, and purifiedby chromatography on SiO₂ (hexanes, 100%, to EtOAc, 100%) to yield 15b(83.0 mg, 0.409 mmol, 64% yield) as a colorless solid: IR (ATR, neat)3067, 1554, 1360, 1333, 1223, 1147 cm⁻¹; ¹H NMR (CDCl₃, 600 MHz) δ 8.09(s, 1 H), 3.89 (s, 3 H); ¹³C NMR (CDCl₃, 150 MHz) δ 153.3, 152.7, 151.3,146.4, 130.4, 30.4.

N-(Biphenyl-4-ylmethyl)-2-chloro-9-methyl-9H-purin-6-amine (16h). To asolution of 15b (60.0 mg, 0.296 mmol) in n-BuOH (1.0 mL) were added4-phenylbenzylamine (58.6 mg, 0.310 mmol) and triethylamine (48.6 mg,0.476 mmol) under an N₂ atmosphere. The reaction mixture was heatedunder microwave irradiation at 120° C. for 30 min. n-BuOH wasevaporated, and the residue was dissolved in EtOAc and washed withwater. The aqueous phase was further extracted with EtOAc, and thecombined organic layers were dried (MgSO₄) and concentrated to yield acolorless solid. The solid was resuspended (hexanes/Et₂O, 2:1),filtered, triturated (hexanes/Et₂O, 2:1), and dried under high-vacuum toyield 16h (89.0 mg, 0.254 mmol, 86%) as an off-white solid: IR (ATR,neat) 3214, 2387, 1620, 1602, 1482, 1308, 1233 cm⁻¹; ¹H NMR(CDCl₃/CD₃OD, 9/1, 600 MHz) δ 7.64 (bs, 1 H), 7.49 (d, 4 H, J=7.8 Hz),7.38 (d, 2 H, J=7.8 Hz), 7.34 (t, 2 H, J=7.8 Hz), 7.25 (t, 1 H, J=7.8Hz), 4.74 (bs, 2 H), 3.77 (s, 3 H); ¹³C NMR (CDCl₃/CD₃OD, 9/1, 150 MHz)δ 154.7, 154.5, 150.1, 140.5, 140.4, 140.3, 136.7, 128.6, 128.2, 127.1,126.8, 117.8, 44.1, 29.8.

(R)-2-(6-(Biphenyl-4-ylmethylamino)-9-methyl-9H-purin-2-ylamino)butan-1-ol(13h). A mixture of 16h (50.0 mg, 0.143 mmol) and(R)-(−)-2-amino-1-butanol (68.4 mg, 0.721 mmol) was heated in amicrowave vial immersed in an oil bath at 170° C. for 15 h. The reactionmixture was cooled to room temperature, diluted with water, andextracted with EtOAc (3×). The combined organic phases were washed withwarm water (50-55° C., 2×) dried (MgSO₄), concentrated, and dried underhigh-vacuum to yield an oily, yellow-green residue. Addition of Et₂O andhexanes resulted in the precipitation of a light green solid. The solidwas rinsed (Et₂O, 3×) by pipetting out the supernatant and dried underhigh-vacuum at 40° C. to yield impure product (90% purity). The solidand filtrate were combined, preadsorbed onto SiO₂ and purified bychromatography on SiO₂ (hexanes/EtOAc, 1:1, to EtOAc/Et₃N, 99:1, toEtOAc/MeOH/Et₃N, 98:10:1) to yield 13h (35.7 mg, 0.0887 mmol, 62%) as alight green solid: Mp 133-136° C.; IR (ATR, neat) 3290, 2927, 1600, 1487cm⁻¹; ¹H NMR (CDCl₃, 600 MHz) δ 7.59 (d, 2 H, J=7.2 Hz), 7.56 (d, 2 H,J=7.8 Hz), 7.47-7.43 (m, 4 H), 7.41 (s, 1 H), 7.35 (t, 1 H, J=7.2 Hz),6.00 (bs, 1 H), 4.97-4.87 (m, 1 H), 4.83 (bs, 2 H), 4.00-3.90 (m, 1 H),3.83 (dd, 1 H, J=10.8, 2.4 Hz), 3.66 (s, 3 H), 3.64 (dd, 1 H, J=10.8,7.8 Hz), 1.68-1.52 (m, 2 H), 1.03 (t, 3 H, J=7.2 Hz); ¹³C NMR (CDCl₃,150 MHz) δ 160.3, 154.8, 151.0, 140.8, 140.2, 137.8, 137.6, 128.7,128.1, 127.3, 127.0, 114.2, 68.2, 56.2, 44.1, 29.4, 25.0, 10.9; HRMS(EI) m/z calcd for C₂₃H₂₆N₆O 402.2168, found 402.2178.

2-Chloro-N-(2,2-diphenylethyl)-9-methyl-9H-purin-6-amine (16i). To asolution of 15b (68.0 mg, 0.335 mmol) in n-BuOH (1.0 mL) was addedaminodiphenylmethane (66.2 mg, 0.350 mmol) and triethylamine (55.1 mg,0.539 mmol) under an N₂ atmosphere. The reaction mixture was heatedunder microwave irradiation at 120° C. for 30 min. n-BuOH wasevaporated, and the residue was dissolved in EtOAc and washed withwater. The aqueous phase was further extracted with EtOAc, and thecombined organic extracts were dried (MgSO4) and concentrated to yield acolorless solid. The solid was resuspended (hexanes/Et₂O, 2:1),filtered, triturated (hexanes/Et₂O, 2:1), and dried under high-vacuum toyield 16i (83.0 mg, 0.237 mmol, 71%) as an off-white, crude solid thatwas used without further purification: Characteristic signals: ¹H NMR(CDCl₃, 600 MHz) δ 7.40-7.20 (m, 12 H), 6.75 (bs, 1 H), 3.69 (s, 3 H);¹³C NMR (CDCl₃, 150 MHz) δ 154.5, 154.2, 150.7, 141.3, 140.9, 128.5,127.6, 127.4, 118.4, 57.3, 29.9.

(R)-2-(6-(2,2-Diphenylethylamino)-9-methyl-9H-purin-2-ylamino)butan-1-ol(13i). A mixture of 16i (50.0 mg, 0.143 mmol) and(R)-(−)-2-amino-1-butanol (68.4 mg, 0.721 mmol) was heated in amicrowave vial immersed in an oil bath at 170° C. for 15 h. The reactionmixture was cooled to room temperature, diluted with water, andextracted with EtOAc (3×). The combined organic phases were washed withwarm water (50-55° C., 2×) dried (MgSO₄), concentrated, and dried underhigh-vacuum at 50° C. (oil bath) for 2 h to yield an oily green residue.The crude residue was adsorbed onto SiO₂ and purified by chromatographyon SiO₂ (hexanes/EtOAc, 1:1, to EtOAc/Et₃N, 99:1, to EtOAc/MeOH/Et₃N,94:5:1) to yield 13i (36.5 mg, 0.0907 mmol, 63%) as an off-white solid:Mp 130-134° C.; IR (ATR, neat) 3301 (br), 2932, 1597, 1474 cm⁻¹; ¹H NMR(CDCl₃, 600 MHz) δ 7.40-7.20 (m, 10 H), 6.60-6.40 (m, 2 H), 4.83 (d, 1H, J=6.0 Hz), 3.80-3.75 (m, 1 H), 3.70-3.65 (m, 1 H), 3.59 (s, 3 H),3.60-3.50 (m, 1 H), 1.60-1.40 (m, 2 H), 0.94 (t, 3 H, J=7.2 Hz); ¹³C NMR(CDCl₃, 150 MHz) δ 160.1, 153.9, 151.2, 142.1, 137.6, 128.51, 128.48,127.57, 127.55, 127.3, 127.2, 114.1, 67.5, 58.0, 55.8, 29.3, 24.8, 10.8;HRMS (EI) m/z calcd for C₂₃H₂₆N₆O 402.2168, found 402.2170.

2-Chloro-9-methyl-N-phenethyl-9H-purin-6-amine (16j). To a solution of15b (60.0 mg, 0.296 mmol) in n-BuOH (1.0 mL) was added phenethylamine(38.0 mg, 0.312 mmol) and triethylamine (47.9 mg, 0.474 mmol) under anN₂ atmosphere. The reaction mixture was heated under microwaveirradiation at 120° C. for 30 min. n-BuOH was evaporated, and theresidue was dissolved in EtOAc and washed with water. The aqueous phasewas further extracted with EtOAc, and the combined organic extracts weredried (MgSO₄) and concentrated to yield a colorless solid. The solid wasresuspended (hexanes/Et₂O, 3:1), filtered, triturated (hexanes/Et₂O,3:1), and dried under high-vacuum to yield 16j (71.0 mg, 0.247 mmol,83%) as an off-white amorphous solid: IR (ATR, neat) 3233, 1615, 1578,1299, 1231 cm⁻¹; ¹H NMR (CDCl₃ 600 MHz) δ 7.49 (bs, 1 H), 7.27 (d, 2 H,J=7.2 Hz), 7.24-7.18 (m, 3 H), 6.41 (bs, 1 H), 3.93-3.83 (m, 2 H), 3.77(s, 3 H), 2.97 (t, 2 H, J=7.2 Hz); ¹³C NMR (CDCl₃, 150 MHz) δ 155.1,154.6, 150.3, 140.5, 138.7, 128.8, 128.5, 126.4, 118.5, 41.9, 35.5,29.9.

(R)-2-(9-Methyl-6-(phenethylamino)-9H-purin-2-ylamino)butan-1-ol (13j).A mixture of 16j (50.0 mg, 0.174 mmol) and (R)-(−)-2-amino-1-butanol(83.6 mg, 0.882 mmol) was heated in a microwave vial immersed in an oilbath at 170° C. for 15 h. The reaction mixture was cooled to roomtemperature, diluted with water, and extracted with EtOAc (3×). Thecombined organic phases were washed with warm water (50-55° C., 2×),dried (MgSO₄), concentrated, and dried under high-vacuum to yield anoily yellow residue. Addition of Et₂O and a few drops of hexanes to thesolid resulted in the precipitation of an off-white solid. The solid wasrinsed (Et₂O, 3×) by pipetting out the supernatant and dried underhigh-vacuum at 40° C. The solid and the filtrate were combined, adsorbedonto SiO₂, and purified by chromatography on SiO₂ (hexanes/EtOAc, 1:1,to EtOAc/Et₃N, 99:1, to EtOAc/MeOH/Et₃N, 94:5:1) to yield 13j (37.8 mg,0.111 mmol, 64%) as a light green solid: Mp 108-110° C.; IR (ATR, neat)3922 (br), 2931, 1598, 1488 cm⁻¹; ¹H NMR (CDCl₃, 600 MHz) δ 7.39 (s, 1H), 7.32-7.24 (m, 4 H), 7.22 (t, 1 H, J=7.2 Hz), 5.65 (bs, 1 H), 5.13(bs, 1 H), 4.93-4.85 (m, 1 H), 3.97-3.90 (m, 1 H), 3.90-3.75 (m, 3 H),3.673.63 (m, 1 H), 3.63 (s, 3 H), 2.96 (t, 2 H, J=7.2 Hz), 1.70-1.55 (m,2 H), 1.04 (t, 3 H, J=7.2 Hz); ¹³C NMR (CDCl₃, 150 MHz) δ 160.3, 154.9,150.9, 139.0, 137.5, 128.8, 128.6, 126.4, 114.2, 68.3, 56.2, 41.7, 35.9,29.3, 25.0, 10.9; HRMS (EI) m/z calcd for C₁₈H₂₄N₆O 340.2012, found340.2014.

N-Benzyl-2-chloro-9-methyl-9H-purin-6-amine (16k). To a solution of 15b(71.0 mg, 0.350 mmol) in n-BuOH (1.0 mL) were added benzylamine (40.50mg, 0.370 mmol) and triethylamine (56.6 mmol, 0.560 mmol) under an N₂atmosphere. The reaction mixture was heated under microwave irradiationat 120° C. for 30 min. n-BuOH was evaporated, and the residue wasdissolved in EtOAc and washed with water. The aqueous phase was furtherextracted with EtOAc, and the combined organic extracts were dried(MgSO₄) and concentrated to yield a colorless solid. The solid wasresuspended (hexanes/Et₂O, 3:1), filtered, triturated (hexanes/Et₂O,3:1), and dried under high-vacuum to yield 16k (65.5 mg, 0.239 mmol,68%) as a colorless solid: IR (ATR, neat) 2385, 1596, 1572, 1325, 1232cm⁻¹; ¹H NMR (CDCl₃/CD₃OD, 9/1, 600 MHz) δ 7.72 (bs, 1 H), 7.40 (d, 2 H,J=7.2 Hz), 7.35 (t, 2 H, J=7.2 Hz), 7.29 (t, 1 H, J=7.2 Hz), 4.79 (bs, 2H), 3.79 (s, 3 H); ¹³C NMR (CDCl₃/CD₃OD, 9/1, 150 MHz) δ 154.7, 154.5,150.1, 140.5, 137.6, 128.4, 127.7, 127.4, 117.8, 44.4, 29.8.

(R)-2-(6-(Benzylamino)-9-methyl-9H-purin-2-ylamino)butan-1-ol (13k). Amixture of 16k (49.0 mg, 0.179 mmol) and (R)-(−)-2-amino-1-butanol (85.5mg, 0.902 mmol) was heated in a microwave vial immersed in an oil bathat 170° C. for 11 h. The reaction mixture was cooled to roomtemperature, diluted with water, and extracted with EtOAc (3×). Thecombined organic phases were washed with warm water (50-55° C., 2×),dried (MgS0₄), concentrated, and dried under high-vacuum at 50° C. (oilbath) for 2 h to yield a yellow solid. The addition of Et2O to the solidresulted in the precipitation of an off-white solid. The solid wasrinsed (Et20, 3×) by pipetting out the supernatant and dried underhigh-vacuum overnight at 40° C. to yield an off-white solid. The crudemixture was purified by chromatography on Si0₂ (hexanes/EtOAc, 1:1, toEt0Ac/Me0H/Et₃N, 94:5:1, to EtOAc/MeOH/Et3N, 85:14:1) to yield 13k (35.5mg, 0.109 mmol, 61%) as a light yellow solid: Mp 118-120° C.; IR (ATR,neat) 3261 (br), 2958, 1610, 1493 cm⁻¹; ¹H NMR (CDCl₃, 600 MHz) δ7.40-7.25 (m, 6 H), 6.20 (bs, 1 H), 5.00-4.92 (m, 1 H), 4.74 (bs, 2 H),3.95-3.90 (m, 1 H), 3.81 (dd, 1 H, J=10.8, 2.4 Hz), 3.62 (s, 3 H),3.67-3.58 (m, 1 H), 1.67-1.52 (m, 2 H), 1.01 (t, 3 H, J=7.2 Hz); ¹³C NMR(CDCl₃, 150 MHz) δ 160.3, 154.8, 151.0, 138.7, 137.5, 128.5, 127.6,127.2, 114.1, 67.9, 56.0, 44.3, 29.3, 24.9, 10.9; HRMS (EI) m/z calcdfor C₁₇H₂₂N₆O 326.1855, found 326.1843.

2-Chloro-9-methyl-N-phenyl-9H-purin-6-amine (16l). To a solution of 15b(72.0 mg, 0.355 mmol) in n-BuOH (1.0 mL) were added aniline (34.0 mg,0.365 mmol) and triethylamine (57.4 mg, 0.567 mmol) under an N₂atmosphere. The reaction mixture was heated under microwave irradiationat 120° C. for 30 min. n-BuOH was evaporated, and the residue wasdissolved in EtOAc and washed with water. The aqueous phase was furtherextracted with EtOAc, and the combined organic extracts were dried(MgSO₄) and concentrated to yield a colorless solid. The solid wasresuspended (hexanes/Et₂O, 3:1), filtered, triturated (hexanes/Et₂O,3:1), and dried under high-vacuum to yield 16l (59.7 mg, 0.230 mmol,65%) as a colorless amorphous solid: IR (ATR, neat) 3286, 1620, 1574,1437, 1308, 1236 cm⁻¹; ¹H NMR (CDCl₃, 600 MHz) δ 7.85 (bs, 1 H),7.80-7.70 (m, 3 H), 7.45-7.35 (m, 2 H), 7.20-7.10 (m, 1 H), 3.84 (s, 3H); ¹³C NMR (CDCl₃, 150 MHz) δ 154.2, 152.3, 151.0, 141.4, 137.9, 129.1,124.0, 120.2, 119.1, 30.1.

(R)-2-(9-Methyl-6-(phenylamino)-9H-purin-2-ylamino)butan-1-ol (13l). Amixture of 16l (46.0 mg, 0.177 mmol) and (R)-(−)-2-amino-1-butanol (85.5mg, 0.902 mmol) was heated in a microwave vial immersed in an oil bathat 170° C. for 11 h. The reaction mixture was cooled to roomtemperature, diluted with water, and extracted with EtOAc (3×). Thecombined organic phases were washed with warm water (50-55° C., 2×)dried (MgSO₄), concentrated, and dried under high-vacuum to yield alight green solid. The solid was preadsorbed on SiO₂ and purified bychromatography on SiO₂ (hexanes/EtOAc, 1:1, to EtOAc/Et₃N, 99:1, toEtOAc/MeOH/Et₃N, 90:9:1) to yield 13l (29.8 mg, 0.0954 mmol, 54%) as anoff-white, slightly light green solid: Mp 202-206° C.; IR (ATR, neat)3222, 3133 (br), 2930, 1579, 1498, 1442 cm⁻¹; ¹H NMR (CDCl₃/CD₃OD, 9/1,600 MHz) δ 7.66 (dd, 2 H, J=7.2, 1.2 Hz), 7.43 (s, 1 H), 7.25 (t, 2 H,J=7.2 Hz), 6.97 (td, 1 H, J=7.2, 1.2 Hz), 3.90 (s, 3 H), 3.92-3.85 (m, 1H), 3.67 (dd, 1 H, J=10.8, 3.6 Hz), 3.57-3.53 (m, 1 H), 1.65-1.50 (m, 2H), 0.92 (t, 3 H, J=7.2 Hz); ¹³C NMR (CDCl₃/CD₃OD, 9/1, 150 MHz) δ159.6, 152.1, 151.1, 138.9, 137.79, 137.78, 128.6, 122.9, 119.9, 113.5,65.2, 55.0, 29.1, 24.3, 10.4; HRMS (EI) m/z calcd for C₁₆H₂₀N₆O312.1699, found 312.1693.

2-Chloro-N-(cydopropylmethyl)-9-methyl-9H-purin-6-amine (16m). To asolution of 15b (70.0 mg, 0.345 mmol) in n-BuOH (1.0 mL) were addedaminomethyl-cyclopropane (30.0 mg, 0.4092 mmol) and triethylamine (55.9mg, 0.552 mmol) under an N₂ atmosphere. The reaction mixture was heatedunder microwave irradiation at 120° C. for 30 min. n-BuOH wasevaporated, and the residue was dissolved in EtOAc and washed withwater. The aqueous phase was further extracted with EtOAc, and thecombined organic extracts were dried (MgSO₄), concentrated, and driedunder high-vacuum overnight to yield 16m (77.9 mg, 0.328 mmol, 95%yield) as a colorless solid: IR (ATR, neat) 3260, 1618, 1581, 1301, 1234cm⁻¹; ¹H NMR (CDCl₃, 600 MHz) δ 7.68 (s, 1 H), 6.07 (bs, 1 H), 3.78 (s,3 H), 3.50-3.40 (m, 2 H), 1.13-1.07 (m, 1 H), 0.55 (dd, 2H; J=12.6, 4.8Hz), 0.30 (dd, 2 H, J=9.6, 4.8 Hz); ¹³C NMR (CDCl₃, 150 MHz) δ 155.1,154.6, 150.3, 140.5, 118.5, 45.8, 30.0, 10.5, 3.5.

(R)-2-(6-(Cyclopropylmethylamino)-9-methyl-9H-purin-2-ylamino)butan-1-ol(13m). A mixture of 16m (50.0 mg, 0.210 mmol) and(R)-(−)-2-amino-1-butanol (95.0 mg, 1.00 mmol) was heated in a microwavevial immersed in an oil bath at 170° C. for 11 h. The reaction wascooled to room temperature, diluted with water, and extracted with EtOAc(3×). The combined organic phases were washed with warm water (50-55°C., 2×), dried (MgSO₄), concentrated, and dried under high-vacuum toyield a green solid. Addition of Et₂O to the solid resulted in theprecipitation of an off-white solid. The solid was rinsed (Et₂O, 3×) bypipetting out the supernatant and dried under high-vacuum to yield alight green solid. The crude residue was purified by chromatography onSiO₂ (hexanes/EtOAc, 1:1, to EtOAc/MeOH/Et₃N, 94:5:1, toEtOAc/MeOH/Et₃N, 84:15:1) to yield 13m (35.8 mg, 0.123 mmol, 59%) as acrystalline green solid: Mp 147-150° C.; IR (ATR, neat) 3328, 3078,2849, 1610, 1490 cm⁻¹; ¹H NMR (CDCl₃, 600 MHz) δ 7.40 (s, 1 H), 5.83(bs, 1 H), 5.37 (bs, 1 H), 5.00-4.87 (m, 1 H), 3.95-3.88 (m, 1 H), 3.82(d, 1 H, J=10.8 Hz), 3.67-3.58 (m, 1 H), 3.62 (s, 3 H), 3.39 (bs, 2 H),1.68-1.50 (m, 2 H), 1.131.03 (m, 1 H), 1.02 (t, 3 H, J=7.2 Hz),0.58-0.48 (m, 2 H), 0.31-0.21 (m, 2 H); ¹³C NMR (CDCl₃, 150 MHz) δ160.3, 154.8, 150.8, 137.3, 114.0, 68.1, 56.1, 45.4, 29.3, 25.0, 10.9,10.8, 3.4; HRMS (EI) m/z calcd for C₁₄H₂₂N₆O 290.1855, found 290.1850.

2-Chloro-9-methyl-N-(pyridin-3-ylmethyl)-9H-purin-6-amine (16n). To asolution of 15b (72.0 mg, 0.355 mmol) in n-BuOH (1.0 mL) was added3-(aminomethyl)pyridine (39.7 mg, 0.367 mmol) and triethylamine (57.5mg, 0.568 mmol) under an N₂ atmosphere. The reaction mixture was heatedunder microwave irradiation at 120° C. for 30 min. n-BuOH wasevaporated, and the residue was dissolved in EtOAc and washed withwater. The aqueous phase was further extracted with EtOAc, and thecombined organic extracts were dried (MgSO₄) and concentrated to yield acolorless solid. The solid was resuspended (hexanes/Et₂O, 1:1),filtered, triturated (hexanes/Et₂O, 1:1), and dried under high-vacuum toyield 16n (76.0 mg, 0.277 mmol, 78%) contaminated with a small amount(˜10%) of EtOAc and Et₂O as a fine yellow amorphous solid that was usedfor the next reaction without further purification: IR (ATR, neat) 3076,2401, 1603, 1579, 1313, 1232 cm⁻¹; ¹H NMR (CDCl₃/CD₃OD, 9/1, 600 MHz) δ8.50 (bs, 1 H), 8.35 (bs, 1 H), 7.71 (d, 1 H, J=7.8 Hz), 7.65 (s, 1 H),7.25-7.20 (m, 1 H), 4.70 (bs, 2 H), 3.68 (s, 3 H); ¹³C NMR (CDCl₃/CD₃OD,9/1, 150 MHz) δ 154.6, 154.3, 148.6, 147.9, 140.7, 136.2, 134.1, 123.6,117.8, 41.6, 29.8.

(R)-2-(9-Methyl-6-(pyridin-3-ylmethylamino)-9H-purin-2-ylamino)butan-1-ol(13n). A mixture of 16n (70.0 mg, 0.255 mmol) and(R)-(−)-2-amino-1-butanol (124 mg, 1.30 mmol) was heated in a microwavevial immersed in an oil bath at 170° C. for 8.5 h. The reaction mixturewas cooled to room temperature, diluted with water, and extracted withEtOAc (3×). The combined organic phases were washed with warmed water(50-55° C., 2×) dried (MgSO₄), concentrated, and dried under high-vacuumto yield an oily, green solid. Addition of Et₂O and a few drops ofhexanes to the solid resulted in the precipitation of a dark greensolid. The solid was carefully crushed with a glass rod, rinsed (Et₂O,3×) by pipetting out the supernatant, and dried under high-vacuumovernight to yield a light green solid. The crude residue was purifiedby chromatography on SiO₂ (hexanes/EtOAc, 1:1, to EtOAc/MeOH/Et₃N,78:20:2) to yield 16n (32.6 mg, 0.0936 mmol, 37%) as a green-gray solid:Mp 136-139° C.; IR (ATR, neat) 3256 (br), 2930, 1603, 1551, 1477 cm⁻¹;¹H NMR (CDCl₃, 600 MHz) δ 8.59 (d, 1 H, J=1.8 Hz), 8.47 (dd, 1 H, J=4.8,1.8 Hz), 7.65 (d, 1 H, J=7.8 Hz), 7.36 (s, 1 H), 7.20 (dd, 1 H, J=7.8,4.8 Hz), 6.58 (bs, 1 H), 5.08-5.00 (m, 1 H), 4.72 (bs, 2 H), 3.94-3.88(m, 1 H), 3.76 (dd, 1 H, J=10.8, 2.4 Hz), 3.61 (s, 3 H), 3.59 (dd, 1 H,J=10.8, 7.2 Hz), 1.66-1.59 (m, 1 H), 1.55-1.49 (m, 1 H), 0.98 (t, 3 H,J=7.2 Hz); ¹³C NMR (CDCl₃, 150 MHz) δ 160.0, 154.5, 151.1, 149.2, 148.5,137.7, 135.3, 134.5, 123.4, 113.9, 67.3, 55.9. 41.8, 29.4, 24.9, 10.8;IR (ATR, neat) 3256 (br), 2930, 1603, 1551, 1477 cm⁻¹; HRMS (EI) m/zcalcd for C₁₆H₂₁N₇O 327.1808, found 327.1806.

2-Chloro-9-propyl-W-(4-(trifluoromethyl)benzyl)-9H-purin-6-amine (16o).To a solution of 15a (80.0 mg, 0.346 mmol) in n-BuOH (1.0 mL) were added4-(trifluoromethyl)benzylamine (63.9 mg, 0.358 mmol) and triethylamine(55.9 mg, 0.552 mmol) under an N₂ atmosphere. The reaction mixture washeated under microwave irradiation at 120° C. for 30 min. n-BuOH wasevaporated, and the residue was dissolved in EtOAc and washed withwater. The aqueous phase was further extracted with EtOAc, and thecombined organic extracts were dried (MgSO₄), and concentrated to yielda colorless solid. The solid was resuspended (hexanes/Et₂O, 3:1),filtered, triturated (hexanes/Et₂O, 3:1), and dried under high-vacuum toyield 16o (78.0 mg, 0.211 mmol, 61%) as a colorless amorphous solid: IR(ATR, neat) 3261, 1630, 1580, 1325, 1308, 1253 cm⁻¹; ¹H NMR (CDCl₃, 600MHz) δ 7.58 (d, 2 H, J=7.8 Hz), 7.52-7.45 (m, 3 H), 6.96 (bs, 1 H), 4.90(bs, 2 H), 4.09 (t, 2 H, J=7.2 Hz), 1.87 (sext, 2 H, J=7.2 Hz), 0.94 (t,3 H, J=7.2 Hz); ¹³C NMR (CDCl₃, 150 MHz) δ 155.0, 154.4, 150.4, 142.3,140.3, 129.8 (q, J=33 Hz), 128.0, 125.5 (q, J=3 Hz), 124.0 (q, J=270Hz), 118.6, 45.5, 43.9, 23.3, 11.0.

(R)-2-(9-Propyl-6-(4-(trifluoromethyl)benzylamino)-9H-purin-2-ylamino)butan-1-ol(130). A mixture of 16o (50.0 mg, 0.135 mmol) and(R)-(−)-2-amino-1-butanol (71.3 mg, 0.751 mmol) was heated in amicrowave vial immersed in an oil bath at 170° C. for 12 h. The reactionmixture was cooled to room temperature, diluted with water, andextracted with EtOAc (3×). The combined organic phases were washed withwarm water (50-55° C., 2×), dried (MgSO₄), concentrated, and dried underhigh-vacuum to yield a yellow solid. Addition of Et₂O/hexanes (1:1) tothe solid resulted in the precipitation of a light green solid. Thesolid was rinsed (Et₂O/hexanes, 1:1) by pipetting out the supernatantand dried under high-vacuum overnight at 40° C. to yield 13o (38.3 mg,0.0907 mmol, 67%) as a light green crystalline solid: Mp 124-127° C.; IR(ATR, neat) 3266, 2962, 1600, 1545, 1326, 1104 cm⁻¹; ¹H NMR (CDCl₃, 600MHz) δ 7.56 (d, 2 H, J=8.4 Hz), 7.46 (d, 2 H, J=7.8 Hz), 7.38 (s, 1 H),6.40 (bs, 1 H), 4.91 (d, 1 H, J=6.0 Hz), 4.95-4.75 (m, 2 H), 3.95 (t, 2H, J=7.2 Hz), 3.89-3.82 (m, 1 H), 3.80 (dd, 1 H, J=10.8, 1.8 Hz), 3.61(dd, 1 H, J=10.8, 7.8 Hz), 1.85 (sextet, 2 H, J=7.2 Hz), 1.64-1.49 (m, 2H), 0.99 (t, 3 H, J=7.2 Hz), 0.94 (t, 3 H, J=7.2 Hz); ¹³C NMR (CDCl₃,150 MHz) δ 160.1, 154.7, 150.8, 143.2, 137.3, 129.4 (q, J=33 Hz), 127.7,125.4 (q, J=3 Hz), 124.1 (q, J=270 Hz), 114.3, 68.2, 56.2, 45.1, 43.8,24.9, 23.2, 11.2, 10.9; HRMS (EI) m/z calcd for C₂₀H₂₅F₃N₆O 422.2042,found 422.2038.

2,6-Dichloro-9-isopropyl-9H-purine (15c). To a solution of2,6-dichloro-9H-purine (0.500 g, 2.65 mmol) in anhydrous DMSO (3.0 mL)cooled to 15° C. was added K₂CO₃ (1.10 g, 7.96 mmol) followed by2-iodopropane (1.35 mL, 13.4 mmol). The mixture was stirred overnight atroom temperature, quenched with water and extracted with EtOAc. Theorganic layers were combined, washed with brine, dried (MgSO₄),concentrated, and purified by chromatography on SiO₂ (hexanes, 100%, tohexanes/EtOAc, 1:1) to yield 15c (0.415 g, 1.80 mmol, 68%) as acolorless solid: Mp 149-151° C.; IR (ATR, neat) 1587, 1554, 1356, 1214cm⁻¹; ¹H NMR (CDCl₃, 600 MHz) δ 8.18 (s, 1 H), 4.92 (hept, 1 H, J=6.6Hz), 1.65 (d, 6 H, J=6.6 Hz); ¹³C NMR (CDCl₃, 150 MHz) δ 152.7, 152.6,151.6, 143.5, 131.0, 48.3, 22.5; HRMS (EI) m/z calcd forC₈H₈Cl₂N₄230.0126, found 230.0120.

2-Chloro-N-(cyclopropylmethyl)-9-isopropyl-9H-purin-6-arnine (16p). To asolution of 15c (100 mg, 0.433 mmol) in n-BuOH (1.5 mL) were addedcyclopropylmethanamine (36.9 mg, 0.519 mmol) and Et₃N (70.9 mg, 0.692mmol). The reaction was heated under microwave irradiation at 120° C.for 20 min. n-BuOH was evaporated in vacuo. The residue was diluted withwater (5.0 mL), and the mixture was extracted with EtOAc (3×7.0 mL). Thecombined organic extracts were dried (MgSO₄), filtered, and concentratedto yield a pale yellow solid. The residue was resuspended (hexanes/Et₂O,2:1), filtered, and washed (hexanes/Et₂O, 3:1). The solid was filteredand dried under high-vacuum to yield 16p (60.3 mg, 0.227 mmol, 52%) as apale yellow solid: Mp 70.2-72.7° C.; IR (ATR) 3286, 3086, 3068, 3055,3038, 3030, 1647, 1627, 1592, 1575, 1560, 1446, 1314, 1273, 1204, 1174,1159, 1150, 1075, 1027, 997, 943, 936, 917, 865, 813, 764, 719, 701, 691cm⁻¹; ¹H NMR (CDCl₃, 300 MHz) δ 7.80 (s, 1 H), 6.03 (bs, 1 H), 4.88-4.79(hept, 1 H, J=6.9 Hz), 3.49 (bs, 2 H), 1.58 (d, 6 H, J=6.9 Hz),1.20-1.10 (m, 1 H), 0.59 (q, 2 H, J=5.7 Hz), 0.32 (q, 2 H, J=4.5 Hz);¹³C NMR (CDCl₃, 75 MHz) δ 155.2, 137.4, 118.9, 46.9, 45.9, 29.7, 22.8,10.7, 3.6; EIMS m/z 265 (M+, 71), 238 (63), 236 (91), 230 (86), 194(72), 182 (57), 86 (94), 84 (100); HRMS (EI) m/z calcd for C₁₂H₁₆ClN₅265.1094, found 265.1096.

(R)-2-(6-(Cyclopropylmethylamino)-9-isopropyl-9H-purin-2-ylamino)butan-1-ol(13p). A mixture of 16p (50.0 mg, 0.188 mmol) and(R)-(−)-2-amino-1-butanol (124.0 mg, 1.40 mmol) was heated in amicrowave vial immersed in an oil bath at 170° C. for 15 h. The reactionmixture was cooled to room temperature, diluted with water (7.0 mL), andextracted with EtOAc (4×10.0 mL). The combined organic phases werewashed with warm water (50-55° C., 2×5.0 mL), dried (MgSO₄),concentrated, and dried under high-vacuum overnight to yield a yellowoil. The yellow oil was dissolved in EtOAc and suspended in Et₂O.Dropwise addition of hexanes (minimal solvent added to achieve ahomogeneous supernatant) precipitated an off-white solid. The solid wasrinsed (Et₂O/hexanes, 2:1) by pipetting out the supernatant and dried toobtain crude 13p (51.0 mg, 0.160 mmol, 85%) as an oil that was usedwithout further purification: IR (ATR) 3286, 3086, 3068, 3055, 3038,3030, 1647, 1627, 1592, 1575, 1560, 1446, 1314, 1273, 1204, 1174, 1159,1150, 1075, 1027, 997, 943, 936, 917, 865, 813, 764, 719, 700, 691 cm⁻¹;¹H NMR (CDCl₃, 300 MHz) δ 7.52 (s, 1 H), 5.85 (bs, 1 H), 4.91 (bs, 1 H),4.58 (hept, 1 H, J=6.3 Hz), 3.92-3.80 (m, 2 H), 3.72-3.60 (m, 1 H),3.60-3.30 (m, 2 H), 1.53 (d, 6 H, J=5.7 Hz), 1.40-0.90 (m, 4 H), 1.03(t, 3 H, J=6.3 Hz), 0.57-0.52 (m, 2 H), 0.28 (bs, 2 H); ¹³C NMR (CDCl₃,75 MHz) δ 160.1, 154.9, 134.4, 119.0, 114.6, 68.7, 56.4, 46.4, 29.7,25.1, 22.6, 11.0, 3.5; EIMS m/z 318 (M⁺, 46), 288 (54), 287 (100), 265(36), 236 (82), 230 (71), 194 (41), 134 (46), 119 (32); HRMS (EI) m/zcalcd for C₁₆H₂₆N₆O 318.2168, found 318.2164.

2-Chloro-9-isopropyl-N-phenethyl-9H-purin-6-amine (16q). To a solutionof 15c (100 mg, 0.433 mmol) in n-BuOH (1.5 mL) were added2-phenylethylamine (62.9 mg, 0.519 mmol) and Et₃N (70.8 mg, 0.692 mmol).The reaction mixture was heated under microwave irradiation at 120° C.for 60 min. n-BuOH was evaporated in vacuo, the residue was diluted withwater, and extracted with EtOAc (3×7.0 mL). The combined organicextracts were dried (MgSO₄) and concentrated to yield a pale yellowsolid. The solid was resuspended (hexanes/Et₂O, 2:1), filtered, andsubsequently rinsed (hexanes/Et₂O, 3:1). The filtered solid was driedunder high-vacuum to yield 16q (112 mg, 0.353 mmol, 82%) as a paleyellow solid: Mp 146.7-148.7° C.; IR (ATR) 3252, 3217, 3211, 3205, 3123,2974, 1616, 1580, 1569, 1457, 1444, 1347, 1308, 1292, 1221, 1198, 1059,745 cm⁻¹; ¹H NMR (CDCl₃, 300 MHz) δ 7.75 (bs, 1 H), 7.33-7.21 (m, 5 H),5.99 (bs, 1 H), 4.83 (hept, 1 H, J=6.9 Hz), 3.91 (bs, 2 H), 3.00 (t, 2H, J=7.2 Hz), 1.58 (d, 6 H, J=6.6 Hz); ¹³C NMR (CDCl₃, 75 MHz) δ 155.3,149.7, 138.8, 137.5, 128.9, 128.6, 126.5, 118.9, 46.8, 42.0, 35.6, 22.9;EIMS m/z 315 (M⁺, 82), 337 (23), 226 (93), 213 (83), 169 (84), 146 (93),119 (100), 104 (83), 77 (87), 65 (81); HRMS (EI) m/z calcd forC₁₆H₁₈ClN₅ 315.1251, found 315.1244.

(R)-2-(9-Isopropyl-6-(phenethylamino)-9H-purin-2-ylamino)butan-1-ol(13q). A mixture of 16q (51.0 mg, 0.161 mmol) and(R)-(−)-2-amino-1-butanol (72.0 mg, 0.792 mmol) was heated in amicrowave vial immersed in an oil bath at 170° C. for 15 h. The reactionmixture was cooled to room temperature, diluted with water, andextracted with EtOAc (4×10.0 mL). The combined organic layers werewashed with warm water (50-55° C., 2×5 mL), dried (MgSO₄), filtered,concentrated, and dried under high-vacuum at 70° C. (oil bath) for 2 hto yield an oily, yellow residue. The crude residue was purified bychromatography on SiO₂ (hexanes/EtOAc, 1:1, to EtOAc/MeOH/Et₃N, 84:5:1)to yield 13q (20.4 mg, 0.0554 mmol, 34%) as a light yellow oil: IR (ATR)3252, 3217, 3211, 3205, 3123, 2974, 1616, 1580, 1569, 1457, 1444, 1347,1308, 1292, 1220, 1198, 1059, 745, 727 cm⁻¹; ¹H NMR (CDCl₃, 300 MHz) δ7.49 (s, 1 H), 7.34-7.20 (m, 5 H), 5.76 (bs, 1 H), 4.90 (d, 1 H, J=5.7Hz), 4.63 (hept, 1 H, J=6.9 Hz), 3.95-3.80 (m, 4 H), 3.66 (dd, 2 H,J=7.8, 10.5 Hz), 2.97 (t, 2 H, J=7.2 Hz), 1.70-1.50 (m, 2 H), 1.53 (d, 6H, J=6.6 Hz), 1.05 (t, 3 H, J=7.5 Hz); ¹³C NMR (CDCl₃, 75 MHz) δ 160.4,155.2, 139.3, 134.7, 129.1, 128.9, 126.7, 115.0, 69.0, 56.7, 46.6, 42.1,36.3, 25.3, 22.9, 11.3; EIMS m/z 368 (M⁺, 84), 338 (77), 277 (43), 205(77), 163 (77), 105 (85), 91 (100); HRMS (EI) m/z calcd for C₂₀H₂₈N₆O368.2325, found 368.2308.

2-Chloro-9-isopropyl-N-(pyridin-3-ylmethyl)-9H-purin-6-amine (16r). To asolution of 15c (99.0 mg, 0.423 mmol) in n-BuOH (1.5 mL) were added3-pyridinemethanamine (55.6 mg, 0.514 mmol) and Et₃N (70.1 mg, 0.685mmol). The reaction mixture was heated under microwave irradiation at120° C. for 20 min. n-BuOH was evaporated in vacuo. The residue wasdiluted with water (5.0 mL) and extracted with EtOAc (3×7.0 mL). Thecombined organic extracts were dried (MgSO₄), filtered, and concentratedto yield a pale yellow solid. The solid residue was resuspended(hexanes/Et₂O, 2:1), filtered, and the solid was rinsed (hexanes/Et₂O,3:1). The filtered solid was dried under high-vacuum to yield 16r (112.0mg, 0.389 mmol, 86%) as a pale yellow solid: ¹H NMR (CDCl₃, 300 MHz) δ8.65 (s, 1 H), 8.54 (dd, 2 H, J=1.2, 4.5 Hz), 7.73-7.70 (m, 1 H),7.28-7.23 (m, 1 H), 6.62 (bs, 1 H), 5.00-4.75 (m, 2 H), 4.83 (hept, 1 H,J=6.9 Hz), 1.57 (d, 6 H, J=6.6 Hz).

(R)-2-(9-Isopropyl-6-(pyridin-3-ylmethylamino)-9H-purin-2-ylamino)butan-1-ol(13r). A mixture of 16r (50.0 mg, 0.165 mmol) and(R)-(−)-2-amino-1-butanol (73.6 mg, 0.826 mmol) was heated in amicrowave vial immersed in an oil bath at 170° C. for 15 h. The reactionmixture was cooled to room temperature, diluted with water (5.0 mL), andextracted with EtOAc (4×10 mL). The combined organic phases were washedwith warm water (50-55° C., 2×5 mL), dried (MgSO₄), filtered,concentrated, and dried under high-vacuum overnight to yield a yellowoil. The oil was dissolved in EtOAc and Et₂O, and upon drop-wiseaddition of hexanes an off-white solid precipitated. The solid wasrinsed (Et₂O/hexanes, 2:1, 3×) by pipetting out the supernatant. Afterdrying the solid under high-vacuum, 13r (35.9 mg, 0.101 mmol, 61%) wasobtained as a colorless amorphous solid: IR (ATR) 3252, 3217, 3211,3205, 3123, 2974, 1616, 1580, 1569, 1457, 1444, 1347, 1308, 1292, 1221,1198, 1059, 745, 727 cm⁻¹; ¹H NMR (CDCl₃, 300 MHz) δ 8.60 (d, 1 H, J=1.5Hz), 8.48 (dd, 1 H, J=1.2, 4.5 Hz), 7.70-7.65 (m, 1 H), 7.507.44 (m, 1H), 7.20 (dd, 1 H, J=4.8 Hz, 7.8 Hz), 6.57 (bs, 1 H), 4.98-4.95 (m, 1H), 4.804.70 (m, 2 H), 4.58 (hept, 1 H, J=6.9 Hz), 3.91-3.86 (m, 1 H),3.77 (dd, 1 H, J=3.0, 7.8 Hz), 3.61 (dd, 2 H, J=7.2, 10.8 Hz), 1.75-1.40(m, 2 H), 1.50 (d, 6 H, J=6.6 Hz), 0.97 (t, 3 H, J=7.5 Hz).

N-(Biphenyl-4-ylmethyl)-2-chloro-9-isopropyl-9H-purin-6-amine (16s). Toa solution of 15c (150.0 mg, 0.649 mmol) in n-BuOH (1.5 mL) were added4-phenylbenzylamine (0.125 0.682 mmol) and triethylamine (108.0 mg, 1.06mmol) under an N₂ atmosphere. The reaction mixture was heated undermicrowave irradiation at 120° C. for 20 min. n-BuOH was evaporated, andthe residue was dissolved in EtOAc and washed with water. The aqueousphase was further extracted with EtOAC, and the combined organicextracts were dried (MgSO₄) and concentrated to yield a colorless solid.The solid was resuspended (hexanes/Et₂O, 3:1), filtered, and rinsed(hexanes/Et₂O, 3:1). The solid was dried under high-vacuum to yield 16s(187.0 mg, 0.495 mmol, 76%) as an off-white solid: Mp 98-100° C.; IR(ATR, neat) 1615, 1571, 1350, 1308 cm⁻¹; ¹H NMR (CDCl₃, 600 MHz) δ 7.68(bs, 1 H), 7.61-7.57 (rm 4 H), 7.487.42 (m, 4 H), 7.36 (L 1H J=7.8 Hz),6.55 (bs, 1 H), 4.88 (bs, 2 H), 4.82 (hept, 1 H, J=6.6 Hz), 1.56 (d, 6H, J=6.6 Hz); ¹³C NMR (CDCl₃, 150 MHz) δ 155.1, 154.3, 149.8, 140.6,137.7, 137.0, 128.8, 128.4, 127.4, 127.3, 127.1, 118.9, 46.9, 44.3,22.8; HRMS (ES) m/z calcd for C₂₁H₂₀ClN₅ [M+Na]⁺400.1305, found400.1308.

(R)-2-(6-(Biphenyl-4-ylmethylamino)-9-isopropyl-9H-purin-2-ylamino)butan-1-ol(13s). A mixture of 16s (32.0 mg, 0.0821 mmol), potassium fluoride (1.50mg, 0.0258 mmol), and (R)-(−)-2-amino-1-butanol (61.8 mg, 0.651 mmol)was heated in a microwave vial immersed in an oil bath at 170° C. for 12h. The reaction mixture was cooled to room temperature, diluted withwater, and extracted with EtOAc (4×). The combined organic phases werewashed with warm water (50-55° C., 2×), dried (MgSO₄), filtered,concentrated, and dried under high-vacuum at 70° C. (oil bath) for 2 hto yield an amorphous yellow semi-solid. After addition of Et₂O, theproduct was precipitated from the solution by drop-wise addition ofhexanes (added in a minimal amount to achieve a homogeneous mixture).The solid was rinsed (Et₂O/hexanes, 2:1) by pipetting out thesupernatant. The solid was dried under high-vacuum at 40° C. overnight(to eliminate a volatile impurity, ˜0.9 ppm) to obtain 13s (25 mg,0.0581 mmol, 71%) as a light yellow solid: Mp 116-119° C.; IR (ATR,neat) 3265, 1600, 1542, 1485 cm⁻¹; ¹H NMR (CDCl₃, 600 MHz) δ 7.60-7.52(m, 4 H), 7.46-7.39 (m, 5 H), 7.34 (t, 1 H, J=7.8 Hz), 6.52 (bs, 1 H),4.97 (s, 1 H), 4.80 (bs, 2 H), 4.59 (hept, 1 H, J=6.6 Hz), 3.96-3.88 (m,1 H), 3.83 (dd, 1 H, J=10.8, 2.4 Hz), 3.51 (dd, 1 H, J=10.8, 7.8 Hz),1.68-1.50 (m, 2 H), 1.51 (d, 6 H, J=6.6 Hz), 1.02 (t, 3 H, J=7.8 Hz);¹³C NMR (CDCl₃, 150 MHz) δ 160.0, 154.8, 150.1, 140.8, 140.2, 138.0,134.5, 128.7, 128.1, 127.3, 127.2, 127.0, 114.6, 68.3, 56.2, 46.4, 44.0,25.0, 22.5, 22.4, 10.9; HRMS (ES) m/z calcd for C₂₅H₃₀N₆O [M+H]⁺431.2559, found 431.2538.

N-Benzhydryl-2-chloro-9-isopropyl-9H-purin-6-amine (16t). To a solutionof 15c (100.0 mg, 0.433 mmol) in n-BuOH (1.5 mL) were addeddiphenylamine (95.2 mg, 0.519 mmol) and Et₃N (70.8 mg, 0.692 mmol). Thereaction mixture was heated under microwave irradiation at 120° C. for20 min. n-BuOH was evaporated in vacuo, and the residue was diluted withwater (5.0 mL), and extracted with EtOAc (3×7.0 mL). The combinedorganic extracts were dried (MgSO4), filtered, and concentrated to yielda pale yellow solid that was resuspended (hexanes/Et₂O, 2:1), filtered,and washed hexanes/Et₂O, 3:1). The filtrate was dried under high-vacuumto obtain 16t (117 mg, 0.310 mmol, 72%) as a pale yellow solid: Mp191.1-193.2° C.; IR (ATR) 3252, 3217, 3211, 3205, 3123, 2974, 1616,1580, 1569, 1457, 1444, 1347, 1308, 1292, 1221, 1198, 1059, 745, 727cm⁻¹; ¹H NMR (CDCl₃, 300 MHz) δ 7.63 (bs, 1H), 7.32-7.26 (m, 10 H), 6.76(bs, 1 H), 4.80 (hept, 1 H, J=6.6 Hz), 1.55 (d, 6 H, J=6.9 Hz); ¹³C NMR(CDCl₃, 75 MHz) δ 154.4, 150.0, 141.5, 137.9, 128.8, 127.7, 127.5,127.2, 118.8, 57.3, 46.9, 22.8; EIMS m/z 377 (M+, 98), 379 (35), 334(25), 182 (44), 167 (100), 165 (61); HRMS (EI) m/z calcd for C₂₁H₂₀ClN₅377.1407, found 377.1400.

(R)-2-(6-(Benzhydrylamino)-9-isopropyl-9H-purin-2-ylamino)butan-1-ol(13t). A mixture of 16t (50.0 mg, 0.132 mmol) and(R)-(−)-2-amino-1-butanol (87.5 mg, 0.981 mmol) was heated in amicrowave vial immersed in an oil bath at 170° C. for 15 h. The reactionmixture was cooled to room temperature, diluted with water (5.0 mL), andextracted with EtOAc (4×10.0 mL). The combined organic layers werewashed with warm water (50-55° C., 2×5.0 mL), dried (MgSO₄), filtered,concentrated, and dried under high-vacuum overnight to obtain a yellowoil. The oil was dissolved in EtOAc, resuspended in Et₂O, and hexaneswas added drop-wise to achieve a homogeneous supernatant. An off-whitesolid precipitated from the solution, and the solid was rinsed(Et₂O/hexanes, 2:1) by pipetting out the supernatant and dried underhigh-vacuum to obtain 13t (36.4 mg, 0.0845 mmol, 64%) as an off-whitesolid: Mp 72.2-75.0° C.; IR (ATR) 3286, 3086, 3068, 3055, 3038, 3030,1647, 1627, 1592, 1575, 1560, 1446, 1314, 1273, 1204, 1174, 1159, 1150,1075, 1027, 997, 943, 936, 917, 865, 813, 764, 719, 701, 691 cm⁻¹; ¹HNMR (CDCl₃, 300 MHz): δ 7.46 (s, 1 H), 7.34-7.26 (m, 10 H), 6.53 (bs, 1H), 6.41 (bs, 1 H), 4.81 (d, 1 H, J=5.1 Hz), 4.59 (hept, 1 H, J=6.6 Hz),3.78-3.70 (m, 2 H), 3.57-3.51 (dd, 2 H, J=7.5, 9.9 Hz), 1.6-1.3 (m, 2H), 1.51 (d, 6 H, J=6.6 Hz), 0.96 (t, 3 H, J=7.5 Hz); ¹³C NMR (CDCl₃, 75MHz) δ 159.9, 153.9, 142.2, 134.6, 128.5, 127.6, 127.3, 114.6, 67.9,57.9, 56.1, 46.4, 24.9, 22.6, 10.9; EIMS m/z 430 (M⁺, 89), 400 (78), 399(100), 358 (36), 168 (63), 165 (91), 152 (59); HRMS (EI) m/z calcd forC₂₅H₃₀N₆O 430.2481, found 430.2486.

2-Chloro-9-isopropyl-N-((5-methylthiophen-2-yl)methyl)-9H-purin-6-amine(16u). To a solution of 15c (74.4 mg, 0.322 mmol) in n-BuOH (1.25 mL)was added (5-methylthien-2-yl)methylamine<<HCl (55.7 mg, 0.340 mmol) andfreshly distilled Et₃N (98.0 mg, 0.969 mmol) under an N₂ atmosphere. Thereaction mixture was subjected to microwave irradiation at 120° C. for30 min. White crystals were observed upon completion of the heating.n-BuOH was evaporated in vacuo, and the residue was dissolved in EtOAc(20.0 mL) and deionized water (10.0 mL). The aqueous phase was furtherextracted with EtOAc (3×10.0 mL), and the combined organic layers weredried (MgSO₄), filtered, and concentrated to yield a light yellow solid.The solid was resuspended (hexanes/Et₂O, 3:1), and the precipitatedsolid was filtered through a fritted funnel and dried under high-vacuumovernight to obtain 16u (96.9 mg, 0.301 mmol, 94%) as light yellowamorphous solid: IR (ATR, neat) 3340, 3256, 3213, 3184, 3137, 3120,3064, 2977, 2967, 2921, 1705, 1676, 1620, 1569, 1538, 1463, 1351, 1310,1290, 1256, 1224, 1200, 1159, 1098, 1070, 1036, 1010, 969, 956, 798,787, 761, 736, 725, 695, 678, 658 cm⁻¹; ¹H NMR (300 MHz, CDCl₃) 7.77(bs, 1 H), 6.85 (d, 1 H, J=3.3 Hz), 6.60 (d, 1 H, J=3.3 Hz), 6.25 (bs, 1H), 4.89 (bs, 2 H), 4.83 (sept, 1 H, J=6.7 Hz), 2.45 (s, 3 H), 1.58 (d,6 H, J=6.6 Hz); ¹³C NMR (75 MHz, CDCl₃) δ 154.8, 154.2, 149.9, 139.9,138.5, 137.8, 126.0, 124.7, 118.8, 46.9, 39.6, 22.8, 15.4; HRMS (ES) m/zcalcd for C₁₄H₁₆N₅SCl 321.0850, found 321.0813.

(R)-2-(9-Isopropyl-6-((5-methylthiophen-2-yl)methylamino)-9H-purin-2-ylamino)butan-1-ol(13u). A mixture of 16u (65.3 mg, 0.203 mmol) and (R)-2-aminobutan-1-ol(96.2 mg, 101 μL, 1.01 mmol, 5 equiv) were heated in a microwave vialimmersed in an oil bath at 170° C. for 8 h. The reaction mixture wascooled to room temperature, diluted with water (15 mL), and extractedwith EtOAc (3×20.0 mL). The combined organic phases were washed withwarm water (2×10.0 mL, 50-55° C.), dried (MgSO₄), filtered,concentrated, and dried under high-vacuum at 50° C. (oil bath) for 2 hto yield a yellow solid. The crude residue was purified bychromatography on SiO₂ (hexanes/EtOAc, 9:1) to yield 13u (61.8 mg, 0.165mmol, 81%) as a light yellow foam: IR (ATR, neat) 3341, 3272, 3121,2964, 2925, 2052, 2185, 1681, 1605, 1544, 1512, 1493, 1456, 1380, 1311,1253, 1202, 1161, 1102, 1042, 1025, 971, 904, 861, 800, 755, 727, 723,694, 675 cm⁻¹; ¹H NMR (400 MHz, CDCl₃) δ 7.47 (s, 1 H), 6.79 (d, 1 H,J=3.6 Hz), 6.58-6.56 (m, 1 H), 5.98 (bs, 1 H), 5.25-5.00 (b, 1 H), 4.90(app d, 1 H, J=5.6 Hz), 4.82 (bs, 2 H), 4.59 (sept, 1 H, J=6.8 Hz),3.96-3.88 (m, 1 H), 3.84 (dd, 1 H, J=10.4, 2.0 Hz), 3.65 (dd, 1 H,J=10.8, 8.0 Hz), 2.42 (s, 3 H), 1.70-1.50 (m, 2 H), 1.52 (d, 6 H, J=6.8Hz), 1.04 (t, 3 H, J=7.6 Hz); ¹³C NMR (100 MHz, CDCl₃) δ 160.0, 154.4,150.4, 139.7, 139.0, 134.6, 125.8, 124.7, 114.7, 68.6, 56.4, 46.5, 39.7,29.7, 25.0, 22.6, 15.4, 11.0; HRMS (ESI) m/z calcd for C₁₈H₂₇N₆OS[M+Na]⁺397.1767, found 397.1787.

2-Chloro-9-isopropyl-N-(3-(trifluoromethyl)benzyl)-9H-purin-6-amine(16v). To a solution of 15c (65.8 mg, 0.273 mmol) in n-BuOH (1.0 mL)were added 3-(trifluoromethyl)benzylamine (52.4 mg, 0.300 mmol) and Et₃N(46.1 mg, 0.456 mmol) under a nitrogen atmosphere. The reaction mixturewas heated with microwave irradiation at 120° C. for 30 min. The n-BuOHwas evaporated, and the residue was dissolved in EtOAc (20.0 mL) andwashed with water (10.0 mL). The aqueous phase was further extractedwith EtOAc (2×10.0 mL), and the combined organic extracts were dried(MgSO₄), filtered, and concentrated to yield a colorless solid. Thesolid was resuspended (hexanes/Et₂O, 3:1), filtered, washed(hexanes/Et₂O, 3:1), and dried under high-vacuum to yield 16v (63.0 mg,0.170 mmol, 60%) as a colorless amorphous solid: IR (ATR, neat) 3250,3150, 2990, 2925, 1625, 1446, 1313, 1230, 1159, 1140, 1099, 980, 930,700 cm⁻¹; ¹H NMR (300 MHz, CDCl₃) δ 7.77 (s, 1 H), 7.63 (s, 1 H), 7.59(d, 1 H, J=7.5 Hz), 7.56 (d, 1 H, J=7.8 Hz), 7.47 (t, 1 H, J=7.5 Hz),6.33 (bs, 1 H), 4.90 (bs, 2 H), 4.83 (sept, 1 H, J=6.8 Hz), 1.58 (d, 6H, J=6.9 Hz); ¹³C NMR (75 MHz, CDCl₃) δ 155.1, 154.2, 150.0, 139.4,137.8, 131.2, 130.9 (q, J=32.2 Hz), 129.3, 124.4, 124.3, 124.0 (q,J=270.1 Hz), 118.8, 47.0, 43.9, 22.7; HRMS [EI] m/z calcd for[C₁₆H₁₅ClF₃N₅] 369.0968, found 369.9640.

(R)-2-(9-Isopropyl-6-(3-(trifluoromethyl)benzylamino)-9H-purin-2-ylamino)butan-1-ol(13v). A mixture of 16v (56.2 mg, 0.152 mmol) and(R)-(−)-2-aminobutan-1-ol (95.0 mg, 1.07 mmol) was heated in a microwavevial immersed in an oil bath at 170° C. for 8 h. The reaction mixturewas cooled to room temperature, diluted with water (10.0 mL), andextracted with EtOAc (2×15.0 mL). The combined organic phases werewashed with warm water (2×5.0 mL, 50-55° C.), dried (MgSO₄), filtered,concentrated, and dried under high-vacuum at 50° C. (oil bathtemperature) for 2 h to yield a yellow solid. After addition of Et₂O, anoff-white solid precipitated. The solid was rinsed (Et₂O, 3×) bypipetting out the supernatant and dried under high-vacuum overnight at40° C. to yield 13v (16.1 mg, 0.0381 mmol, 25%) as a colorless amorphoussolid: Mp 149.9-153.6° C.; IR (ATR, neat) 3254, 3059, 2934, 1621, 1599,1535, 1323, 1260, 1161, 1118, 1062, 797, 701 cm⁻¹; ¹H NMR (300 MHz,CDCl₃) δ 7.77 (s, 1 H), 7.58-7.51 (m, 3 H), 7.44 (t, 1 H, J=7.5 Hz),6.22 (bs, 1 H), 4.89 (d, 1 H, J=5.7 Hz), 4.83 (bs, 2 H), 4.62 (sept, 1H, J=6.8 Hz), 3.95-3.80 (m, 1 H), 3.82 (dd, 1 H, J=2.7, 10.5 Hz), 3.63(dd, 1 H, J=7.5, 10.5 Hz), 1.70-1.40 (m, 2 H), 1.54 (d, 6 H, J=6.9 Hz),1.01 (t, 3 H, J=7.5 Hz); ¹³C NMR (175 MHz, CDCl₃) δ 159.9, 154.6, 150.3,140.0, 134.7, 130.9, 130.8 (q, J=31.5 Hz), 129.0, 124.4 (q, J=3.5 Hz),124.1 (q, J=3.5 Hz), 124.1 (q, J=271.3 Hz), 114.5, 68.2, 56.2, 46.5,43.8, 24.9, 22.5 (2 C), 10.8; HRMS (ES) m/z calcd for C₂₀H₂₆F₃N₆O [M+H]+423.2120, found 423.2103.

2-Chloro-9-methyl-N-[(5-methylthiophen-2-yl)methyl]-9H-purin-6-amine(16w). To a solution of 15b (30.0 mg, 0.148 mmol, 1 eq) in dry n-BuOH(0.6 mL, 0.25 M) were added (5-methylthien-2-yl)methylamine.HCl (25.4mg, 0.155 mmol, 1.05 eq) and freshly distilled triethylamine (44.9 mg,0.443 mmol, 3 eq, 0.06 mL) under nitrogen. The reaction mixture wassubjected to microwave irradiation at 120° C. for 35 min. The n-BuOH wasevaporated, and the residue was dissolved in EtOAc (20 mL) and deionizedwater (10 mL). The layers were separated and the aqueous layer wasfurther extracted with EtOAc (3×10 mL). The combined organic layers weredried (MgSO₄) and concentrated under reduced pressure to yield a yellowsolid. The solid was washed with Et₂O and dried under high-vacuum toyield 16w (37.5 mg, 0.128 mmol, 86%) as a light yellow solid: Mp217.5-219.6° C.; IR (ATR, neat) 3058, 1607, 1575, 1340, 1303, 1232,1094, 917, 796, 693 cm⁻¹; ¹H NMR (400 MHz, DMSO-d₆) δ 8.76 (bs, 1 H),8.11 (s, 1 H), 6.79 (d, 1 H, J=3.3 Hz), 6.60 (app d, 1 H, J=2.2 Hz),4.68 (d, 2 H, J=5.6 Hz), 3.69 (s, 3 H), 2.34 (s, 3 H); ¹³C NMR (100 MHz,DMSO-d₆) δ 154.4, 152.9, 150.4, 142.1, 139.6, 138.4, 125.7, 124.6,118.1, 38.5, 29.6, 14.9; HRMS (ES) m/z calcd for C₁₂H₁₁N₅SCl [M−H]⁺292.0424, found 292.0428.

(2R)-2-[(9-Methyl-6-{[(5-methylthiophen-2-yl)methyl]amino}-9H-purin-2-yl)amino]butan-1-ol(13w). A mixture of 16w (18.2 mg, 0.0620 mmol, 1 eq) and2-amino-1-butanol (27.6 mg, 0.029 mL, 0.276 mmol, 5 eq) was heated in asealed vial in an oil bath at 170° C. for 15 h. The reaction mixture wascooled at room temperature, treated with water (15 mL) and thenextracted with EtOAc (3×20 mL). The combined organic layers were washedwith warm water (2×10 mL, 50-55° C.), dried (MgSO₄), filtered andconcentrated. Purification by chromatography on SiO₂ (EtOAc/MeOH/Et₃N,94:5:1) gave a yellow oil which was dried under high-vacuum at 50° C.for 2 h to yield 13w (20.0 mg, 0.0577 mmol, 93%) as a yellow oil whichsolidified to a dark yellow solid: Mp 56.4-58.3° C.; IR (ATR, neat)2934, 1601, 1545, 1512, 1415, 1215, 785 cm⁻¹; ¹H NMR (400 MHz, CDCl₃) δ7.35 (s, 1 H), 6.77 (d, 1 H, J=3.2 Hz), 6.55 (app d, 1 H, J=2.3 Hz),6.20 (bs, 1 H), 4.97 (d, 1 H, J=6.1 Hz), 4.80 (bs, 2 H), 3.95 (app pent,1 H, J=5.8 Hz), 3.82 (dd, 1 H, J=10.7, 2.6 Hz), 3.63 (dd, 1 H, J=10.7,7.5 Hz), 3.61 (s, 3 H), 2.41 (s, 3 H), 1.66-1.52 (m, 2 H), 1.02 (t, 3 H,J=7.4 Hz); ¹³C NMR (100 MHz, CDCl3) δ 160.2, 154.3, 151.1, 139.5, 139.1,137.6, 125.7, 124.6, 114.1, 67.9, 56.0, 39.5, 29.3, 25.0, 15.3, 10.9;HRMS (ES) m/z calcd for C₁₆H₂₃N₆OS [M+H]⁺ 347.1654, found 347.1659.

2-Chloro-N-[(5-methylthiophen-2-yl)methyl]-9-propyl-9H-purin-6-amine(16x). To a solution of 15a (33.9 mg, 0.147 mmol, 1 eq.) in n-BuOH (0.6mL, 0.25 M) were added (5-methylthien-2-yl)methylamine-HCl (25.2 mg,0.153 mmol, 1.05 eq.) and freshly distilled triethylamine (44.5 mg,0.440 mmol, 3.00 equiv). The reaction mixture was subjected to microwaveirradiation at 120° C. for 30 min. The residue was dissolved in EtOAc(20 mL) and deionized water (10 mL). The aqueous phase was furtherextracted with EtOAc (3×10 mL), and the combined organic layers weredried (MgSO₄), filtered and concentrated under reduced pressure.Purification on SiO₂ (EtOAc/hexanes, 1:1) gave 16x (42.1 mg, 0.131 mmol,89%) as a colorless solid: Mp 150.3-151.4° C.; IR (ATR, neat) 3256,3208, 2994, 2872, 1616, 1573, 1538, 1472, 1303, 1251, 1219, 1085, 811cm⁻¹; ¹H NMR (400 MHz, CDCl₃) δ 7.57 (bs, 1 H), 6.80 (d, 1 H, J=3.3 Hz),6.75 (bs, 1 H), 6.57 (app dd, 1 H, J=3.3, 1.0 Hz), 4.87 (bs, 2 H), 4.09(t, 2 H, J=7.2 Hz), 2.42 (s, 3 H), 1.88 (sext, 2 H, J=7.4 Hz), 0.94 (t,3 H, J=7.4 Hz); ¹³C NMR (100 MHz, CDCl₃) δ 154.8, 154.5, 150.5, 140.4,140.2, 138.1, 126.4, 124.9, 118.7, 45.6, 39.8, 23.5, 15.5, 11.2; HRMS(ES) m/z calcd for C₁₄H₁₇N₅SCl [M+H]⁺ 322.0893, found 322.0890.

(2R)-2-[(6-{[(5-Methylthiophen-2-yl)methyl]amino}-9-propyl-9H-purin-2-yl)amino]butan-1-ol(13x). A mixture of 16x (20.0 mg, 0.0621 mmol, 1 equiv) and(R)-(−)2-amino-1-butanol (27.7 mg, 0.311 mmol, 5 equiv) was heated in asealed vial in an oil bath at 170° C. for 15 h. The reaction mixture wascooled to room temperature and water was added (15 mL). The mixture wasextracted with EtOAc (3×20 mL), dried (MgSO₄), filtered andconcentrated. Purification by chromatography on SiO₂ (EtOAc/MeOH, 98:2)provided 13x (21.2 mg, 0.0566 mmol, 91%) as a colorless solid: Mp124.8-129.6° C.; IR (ATR, neat) 3418, 3374, 3260, 2958, 1605, 1512,1402, 1333, 1215, 798, 796, 783 cm⁻¹; ¹H NMR (400 MHz, CDCl₃) δ 7.40 (s,1 H), 6.79 (d, 1 H, J=3.4 Hz), 6.57-6.56 (m, 1 H), 6.04 (bs, 1 H), 4.96(app d, 1 H, J=5.5 Hz), 4.82 (bs, 2 H), 3.97-3.88 (m, 3 H), 3.83 (dd, 1H, J=10.7, 2.5 Hz), 3.64 (dd, 1 H, J=10.7, 7.7 Hz), 2.42 (s, 3 H), 1.84(sext, 2 H, J=7.2 Hz), 1.70-1.51 (m, 2 H), 1.03 (t, 3 H, J=7.4 Hz), 0.93(t, 3 H, J=7.2 Hz); ¹³C NMR (100 MHz, CDCl₃) δ 160.1, 154.4, 151.0,139.8, 139.1, 137.4, 126.0, 124.8, 114.5, 68.6, 56.5, 45.2, 39.7, 25.2,23.3, 15.5, 11.3, 11.1; HRMS (ES) m/z calcd for C₁₈H₂₇N₆OS [M+H]+375.1967, found 375.1968.

N-Ethoxycarbonyl-N′-(pyrazol-3-yl)thiourea(1). A solution of1H-pyrazol-3-amine (2.731 g, 32.54 mmol, 1.130 eq) in THF (37 mL) wascooled to 0° C. under nitrogen. In a dropwise fashion, ethoxycarbonylisothiocyanate (3.5 mL, 28 mmol, 1.0 eq) was added to the mixture overapproximately 5 min. The reaction mixture was allowed to stir for 1 h at0° C. Solvent was then removed in vacuo to give 1 as a yellow solid(7.20 g, 28.2 mmol, 98%). This compound was used in the next stepwithout further purification: 1H NMR (CDCl3, 300 MHz) δ 1.34 (t, 3 H,J=7.1 Hz), 4.30 (q, 2 H, J=7.1 Hz), 7.04 (d, 2 H, J=2.4 Hz), 7.55 (d, 2H, J=2.4 Hz), 8.28 (s, 1 H), 11.9 (s, 1 H).

2-Thioxo-2,3-dihydropyrazolo[1,5-a][1,3,5]triazin-4(1 H)-one (2). Asolution of 1 (494 mg, 2.30 mmol, 1.00 eq) in 2 N NaOH (4.8 mL, 10 mmol,4.2 eq) was stirred at room temperature for 2 h. The reaction mixturewas cooled in an ice bath and in a dropwise fashion, 2 N H₂SO₄ (6.9 mL,13 mmol, 6.0 eq,) was added. The resulting precipitate was collected byvacuum filtration, washed with water, and dried under vacuum to give 2as an off white powder (337 mg, 2.01 mmol, 87%). This compound was usedin the next step without further purification: ¹H NMR (DMSO-d₆, 300 MHz)δ 2.50 (s, 3 H), 5.88 (d, 1 H, J=1.5 MHz), 7.86 (d, 1 H, J=1.8 MHz),12.78 (broad s, 1 H), 13.44 (broad s, 1 H).

2-(Methylthio)pyrazolo[1,5-a][1,3,5]triazin-4(3 H)-one (3). To asolution of 2 (4.97 g, 24.2 mmol, 1.00 eq.) in EtOH (97 mL) was addedaqueous 2 N NaOH (25 mL, 70 mmol, 2.0 eq.) and methyl iodide (1.51 mL,24.2 mmol, 1.00 eq.). The slurry was stirred at room temperature for 1.5h. The reaction was then filtered and the solid was dissolved in water(246 mL). Then 2 N H2SO4 was added (14 mL, 28 mmol, 1.2 eq.) and theresulting solid filtered, washed with water, and dried under high vacuumat 60° C. for 5 h to give 3 as a white solid (3.15 g, 14.7 mmol, 60%).This compound was used in the next step without further purification: 1HNMR (DMSO-d6, 400 MHz) δ 2.53 (s, 3 H), 6.35 (d, 1 H, J=2.0 Hz), 7.97(d, 1 H, J=1.6 Hz), 12.89 (s, 1 H).

N-Methyl-2-(methylthio)-N-phenylpyrazolo[1,5-a][1,3,5]triazin-4-amine(4). To a flame dried round bottom flask was added 3 (497 mg, 2.73 mmol,1.00 eq.), POCl3 (8.1 mL, 87 mmol, 32 eq.), and DMAP (1181 mg, 9.67mmol, 3.548 eq.).

The mixture was then heated at reflux for 3.5 h. Excess POCl3 wasremoved under reduced pressure and the resulting light brown residuedried under high vacuum overnight. The solid was dissolved indichloromethane (21 mL), placed under nitrogen, and cooled to 0° C.Triethylamine (2.40 mL, 17.2 mmol 6.31 eq.) and N-methylaniline (1.67mL, 14.9 mmol, 5.47 eq.) were added and, after 10 min, the reaction wasallowed to warm to room temperature and stirred for 7 h. The mixture wasthen diluted with dichloromethane (5 mL), washed with water (4×8 mL),extracted once with dichloromethane (10 mL), rinsed with brine (8 mL)and dried (MgSO4). After concentration, the residue was purified bychromatography on silica gel (3:2, hexanes:dichloromethane, thendichloromethane) to give 4 as a off white solid (564 mg, 1.91 mmol,70%): 1H NMR (CDCl3, 400 MHz) δ 2.53, (s, 3 H), 3.73 (s, 3 H), 6.5 (d, 1H, J=2.0 Hz), 7.17-7.19 (m, 2 H), 7.36-7.41 (m, 3 H), 7.64, (d, 1 H,J=2.4 Hz).

1-(4-(Methyl(phenyl)amino)-2-(methylthio)pyrazolo[1,5-a][1,3,5]triazin-8-yl)propan-1-one(5). To an oven dried vial was added 4 (587 mg, 2.07 mmol, 1.00 eq.),propionyl chloride (0.36 mL, 4.1 mmol, 2.0 eq.) and 1M tin(IV) chloridein dichloromethane (10 mL, 10 mmol, 5.0 eq.). The vial was sealed andallowed to heat at 85° C. for 17 h. The reaction mixture was then pouredover crushed ice, diluted with water (10 mL) and dichloromethane (5 mL)and allowed to stir for 15 min. The layers were separated, the aqueouslayers extracted with dichloromethane (10 mL), the combined organicfractions washed with water and brine (10 mL each), and dried (MgSO4).Following concentration, the crude residue was purified bychromatography on silica gel (98:2 dicloromethane:ethyl acetate) to give5 as a white solid (590 mg, 1.892 mmol, 83%): mp=147.6-148.2° C.; 1H NMR(CDCl3, 300 MHz) δ 1.20 (t, 3 H, J=7.4 Hz), 2.59 (s, 3 H), 3.11 (q, 2 H,J=5.5 Hz), 3.74 (s, 3 H), 7.15-7.18 (m, 2 H), 7.35-7.43 (m, 3 H), 8.09(s, 1 H); 13C NMR (CDCl3, 100 MHz) δ 8.32, 14.37, 34.66, 42.42, 108.90,126.11, 127.62, 129.20, 144.18, 145.65, 148.23, 151.07, 170.71, 195.46;IR ν 2934, 1652, 1501 cm−1;HRMS: Calculated [M+H] for C, 16; H, 17; N,5; OS 328.12; found 328.1220.

N-Methyl-2-(methylthio)-N-phenyl-8-propylpyrazolo[1,5-a][1,3,5]triazin-4-amine(6). To a solution of 5 (203 mg, 0.590 mmol, 1.00 eq) in 1:1dichloromethane:ethanol (4 mL) at 0° C. was added LiCl (66 mg, 1.6 mmol,2.7 eq) and NaBH4 (62 mg, 1.6 mmol, 2.7 eq). The reaction was thenallowed to warm to room temperature and stirred for 15 h. A secondportion of NaBH4 then was added (35 mg, 0.93 mmol, 1.6 eq.) and thereaction stirred an additional 5 h. The reaction was quenched with water(0.5 mL) and the organic solvents removed in vacuo. The residue was thendiluted with dichloromethane (4 mL) and water (2 mL), extracted withdichloromethane (4 mL), washed with brine (2 mL), dried (MgSO4) andconcentrated. The residue was then dissolved in dichloromethane (6 mL)and added to a solution of NaBH4 (227 mg, 5.94 mmol, 10.1 eq.) intrifluoroacetic acid (6.3 mL) at 0° C. The reaction was allowed to warmto room temperature and stirred for 4 h. The reaction was quenched with1 M NaOH (10 mL) and diluted with dichloromethane (5 mL). The aqueouslayer was then extracted with dichloromethane (3×5 mL) and the organicfractions rinsed with saturated sodium bicarbonate (2×7 mL), brine (10mL), dried (MgSO4) and concentrated to give 6 as a yellow oil (158 mg,448 mmol, 75%). This compound was used in the next step without furtherpurification: 1H NMR (CDCl3, 300 MHz) δ 0.93 (t, 3 H, J=7.4 Hz),1.59-1.67 (m, 2 H), 2.55-2.60 (m, 5 H), 3.71 (s, 3 H), 7.15-7.18 (m, 2H), 7.31-7.39 (m, 3 H), 7.52 (s, 1 H); 13C NMR (CDCl3, 100 MHz) δ 13.86,14.16, 23.20, 24.50, 42.02, 107.31, 126.06, 126.91, 128.92, 144.78,145.14, 148.27, 164.93; IR ν 2954, 1534, 1508 cm−1; HRMS: Calculated[M+H] for C, 16; H, 19; N, 5; S, 314.14; found 314.1427.

2-(Methylthio)-N-((5-methylthiophen-2-yl)methyl)-8-propylpyrazolo[1,5-a][1,3,5]triazin-4-amine(7). To a flame dried microwave vial was added mf-00521.014 (99 mg, 0.28mmol, 1.0 eq.), triethylamine (0.78 mL, 5.6 mmol, 20 eq.),(5-methylthien-2-yl)methylamine HCl (234 mg, 1.38 mmol, 4.93 eq.), KF(21 mg, 0.36 mmol 1.3 eq), and absolute ethanol (0.93 mL). The vial wasthen sealed and allowed to heat at 130° C. for 36 h. The reactionmixture was then diluted with water (2 mL), extracted withdichloromethane (3×2 mL), washed with brine (2 mL), and dried (MgSO4).Following concentration, purification was performed by chromatography onsilica gel (hexanes:ethyl acetate, 98:2, 95:5) to give 7 as an off whitesolid (47 mg, 0.13 mmol, 46%): mp=84.9-85.3° C.; 1H NMR (CDCl3, 500 MHz)δ 0.96 (t, 3 H, J=7.3 Hz), 1.65-1.72 (m, 2 H), 2.45 (s, 3 H), 2.45-2.63(m, 5 H), 4.86 (d, 2 H, J=5.5 Hz), 6.59 (d, 2 H, J=1.0 Hz), 6.79, (broads, 1 H), 6.59 (m, 1 H), 7.71 (s, 1 H); 13C NMR (CDCl3, 125 MHz) δ 13.86,14.28, 15.31, 23.31, 24.58, 39.43, 108.76, 124.86, 126.85, 136.71,140.58, 145.29, 146.22, 147.16, 166.02; IR ν 3247, 2954, 1618, 1588cm−1; HRMS: Calculated [M+H] for C, 15; H, 19; N, 5; S, 2 334.11; found334.1153.

2-(Methylsulfonyl)-N-((5-methylthiophen-2-yl)methyl)-8-propylpyrazolo[1,5-a][1,3,5]triazin-4-amine(8). To a solution of 7 (47 mg, 0.0 mmol, 1.00 eq.) in acetone (2.2 mL)at 0° C. was added 3.17 M NaHCO3 (0.33 mL, 1.0 mmol, 8.0 eq.) followedby 0.39 M oxone in water (247 mg in 1.0 mL, 0.402 mmol 3.07 eq.). Theslurry was then allowed to stir 16 h at room temperature. The reactionwas quenched with 50% aqueous sodium bisulfite (0.16 mL), diluted withwater (2 mL), extracted with dichloromethane (3×3 mL), washed with brine(3 mL), dried (MgSO4), and concentrated giving 8 as an opaque oil (48mg, 0.12 mmol, 89%). This compound was used in the next step withoutfurther purification.

(R)-2-((4-(((5-Methylthiophen-2-yl)methyl)amino)-8-propylpyrazolo[1,5-a][1,3,5]triazin-2-yl)amino)butan-1-ol(9) (also referred to herein as compound MF-521-17). To a flame driedmicrowave vial was added 8 (47 mg, 0.011 mmol, 1.00 eq.),R-(−)-2-amino-1-butanol (0.056 mL, 0.57 mmol, 5.0 eq.), KF (34 mg,0.0.57 mmol, 5.0 eq.), and 1,4 dioxane (0.31 mL). The vial was sealedand allowed to heat at 140° C. in a sand bath for 12 h. The reaction wasthen treated with water (2 mL), extracted with dichloromethane (2×3 mL),washed with brine (3 mL), dried (MgSO4), and concentrated. The crudemixture was then purified by chromatography on silica gel (3:1dichloromethane:ethyl acetate) to give 9 as an off white solid (25 mg,0.065 mmol, 57%): mp=118.3-120.1° C.; 1H NMR (CDCl3, 300 MHz) δ 0.954(t, 3 H, J=7.4 Hz), 1.05 (t, 3 H, J=7.5 Hz), 1.57-1.68 (m, 4 H), 2.44(s, 3 H), 2.49 (t, 2 H, J=7.4 Hz), 3.68 (dd, 2 H, J=7.2, 10.8 Hz), 3.84(d, 2 H, J=9.6 Hz), 3.95 (broad s, 1 H), 4.78 (d, 2 H, 6.0 Hz), 6.59 (m,1 H), 6.70 (broad s, 1 H), 6.82 (d, 1 H, J=3.3 Hz), 7.59 (s, 1 H); 13CNMR (CDCl3, 125 MHz) δ 10.80, 13.81, 15.29, 23.32, 24.52, 24.83, 39.31,56.26, 67.90, 105.82, 124.82, 126.55, 136.96, 140.40, 145.58, 146.65,148.44, 157.73; IR ν 3286, 2956, 2934, 1592, 1564 cm−1; HRMS: Calculated[M+H] for C, 18; H, 26; N, 6; OS, 375.19; found 375.1960.

EXAMPLE 1 Activity of Analogs 13a-13x

tsA201 cells expressing calcium channels. Biological evaluations of theeffects of (R)-roscovitine derivatives on N-type calcium channels wereinitially performed using a tsA201 cell line that stably expresses allof the subunits of the N-type Ca²⁺ channel splice variant predominantlypresent in mammalian brain and spinal cord: Ca_(v)2.2 rnα_(1B-c) (Ca_(v)2.2 e[24a,Δ31a]), Ca_(v)β₃ and Ca_(v)α₂δ₁. For subsequent evaluation ofeffects on N-, P/Q-, or L-type channels, tsA-201 cells were transientlytransfected with Cav2.2, Cav2.1, or Cav1.3, in combination with Ca_(v)β₃and Ca_(v)α₂β₁ (Addgene, Cambridge Mass.) using FuGENE 6 (LifeTechnologies, Grand Island, N.Y.). All cells were maintained in DMEMsupplemented with 10% fetal bovine serum. For the stable cell lineexpressing N-type channels, 25 μg/mL zeocin, 5 μg/mL blasticidin, and 25μg/mL hygromycin were added as selection agents.

Whole-cell patch clamp recordings of calcium current. To assess thebiological effects of (R)-roscovitine derivatives, whole-cell currentsthrough Ca²⁺ channels were recorded using perforated patch methods.Briefly, the pipette solution consisted of 70 nM Cs₂SO₄, 60 mM CsCl, 1mM MgCl₂, 10 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid(HEPES) at pH 7.4. Cultured cells were bathed in a saline composed of130 mM choline chloride (ChCl), 10 mM tetraethylammonium chloride(TEA-Cl), 2 mM CaCl₂, 1 mM MgCl₂, 10 mM HEPES, at pH 7.4. Patch pipetteswere fabricated from borosilicate glass and pulled to a resistance ofabout 1 MΩ. Before each experiment, a stock solution consisting of 3 mgamphotericin-B dissolved into 50 μL anhydrous DMSO was made. Patchpipettes were tip-dipped into pipette solution that did not containamphotericin-B for 5-10 seconds, and then backfilled with pipettesolution that contained amphotericin-B (7 μL amphotericin-B stocksolution mixed into 500 μL pipette solution, made fresh every hour).Using this approach, perforated patch access resistances were 7.41±1.75MΩ (mean±SD, n=68). Capacitive currents and passive membrane responsesto voltage commands were subtracted from the data. Currents wereamplified by an Axopatch 200B amplifier, filtered at 5 KHz, anddigitized at 10 KHz for subsequent analysis using pClamp software (AxonInstruments/Molecular Devices; Sunnyvale, Calif.). A liquid junctionpotential of −11.3 mV was subtracted during recordings. To measureeffects on calcium channel tail currents, the tail current integral wasmeasured before and after application of a derivative, with the integralof each trace being normalized to its peak. All experiments were carriedout at room temperature (22° C.). All (R)-roscovitine derivatives weredissolved in DMSO as a 100 mM stock and stored at −20° C. For whole-cellrecordings, (R)-roscovitine derivatives were diluted on the day of useinto saline at a final concentration of 1-100 μM and bath applied via aglass pipette in a ˜0.5 mL static bath chamber. Control recordingsperformed with 0.1-1% DMSO alone added to the drug delivery pipettesolution revealed no significant effects on whole cell Ca²⁺ currents.All other salts and chemicals were obtained from Sigma-Aldrich chemicalcompany (St. Louis, Mo.).

Kinase inhibitory activity. Kinase inhibitory activities were determinedusing the EMD Millipore KinaseProfiler™ service (Millipore UK Ltd.).Each compound's kinase inhibitory activity was tested at three differentconcentrations (0.2 μM, 2 μM, and 20 μM) against five different kinases:Cdk1 cyclinB(h), cdk2 cyclinA(h), cdk5 p35(h), mitogen-activated proteinkinase (MAPK1(h)) and myosin light-chain kinase (MLCK(h)).

Results. Compounds 13a-13x were evaluated to determine their N-type Ca²⁺channel agonist and cdk2 kinase inhibitory properties. The results areshown in Table 2. Compound 13d showed a ca. 2-fold increased agonism anda 22-fold decreased cdk2 kinase activity vs. the benchmark,(R)-roscovitine. The decreased kinase activity of 13d was attributed tothe replacement of the i-propyl side chain at R¹ with the more flexiblen-propyl group, a hypothesis that was supported by the similarlydecreased cdk2 activity of 13g. The preference of the R¹ group for thebranched i-propyl group with regard to cdk2 activity is quitepronounced, as shown for the methylated 13k, which also reduced thekinase activity ca. 8-fold. All modifications of the benzyl group at R²,including the cyclopropyl-methyl group in 13g, led to complete loss ofN-type Ca²⁺ channel activity.

The first group at R² that proved to be an effective mimic of the benzylgroup was the methylthiophenyl-substituted 13u. This compound had almost4-fold improved Ca²⁺ channel agonism, even though it was also a lownanomolar cdk2 inhibitor with a 22-fold decreased cdk2 activity comparedto (R)-roscovitine. Since 13u was substituted with i-propyl at R¹, itwas determined that replacing the i-propyl group with a methyl or ann-propyl group might produce reduced kinase inhibitory propertiesanalogous to 13d, 13g and 13k. Indeed, this turned out to be the case.Both 13w and 13x had an EC₅₀=3 μM against cdk2, but the R¹=methylsubstitution in 13w also decreased the channel activity ca. 3-fold vs13u. In contrast, 13x proved to be a considerably more potent Ca²⁺channel agonist with an EC₅₀=7.2 μM for N-type channels. Thus, smallchanges in the R¹ substitution on the calcium channel affinity weresurprisingly effective.

As seen in Table 2, three compounds in particular exhibited a strongagonist effect on the Ca²⁺ channel tail currents: compounds 13u, 13w,and 13x (FIG. 3A). For comparison, the effect of (R)-roscovitine onN-type tail currents was also determined. By measuring the tail currentintegrals (first normalizing each trace to its peak tail currentamplitude and then normalizing to control integrals), the EC₅₀ values of(R)-roscovitine, 13w, 13u and 13x were found to be 27.58±1.65 μM,30.02±1.87 μM, 11.29±1.48 μM and 7.21±0.86 μM, respectively (FIG. 3B).Furthermore, the maximal fold increase in the tail current integralrelative to control was ˜8-fold, ˜13-fold, ˜25-fold and ˜32-fold, whenmodified by (R)-roscovitine, 13w, 13u and 13x, respectively.

TABLE 2 N-Type Ca²⁺ Channel Activity Cdk2 Activity Compound EC₅₀ +/− SEM[μM]^(a) EC₅₀ +/− SEM [μM]^(b) (R)-roscovitine 27.58 +/− 1.65  0.151 +/−0.004 13a >100 ND 13b >100 ND 13c >100 ND 13d 14.23 +/− 2.71  3.34 +/−0.05 13e >100 ND 13f >100 ND 13g >100 3.63 +/− 0.42 13h >100 ND 13i >100ND 13j >100 ND 13k >70 1.44 +/− 0.02 13l >100 ND 13m >100 ND 13n >100 ND13o >100 ND 13p >100 ND 13q >100 ND 13r >100 ND 13s >100 ND 13t >100 ND13u 11.29 +/− 1.48   0.262 +/− 0.0002 13v >100 ND 13w 30.02 +/− 1.87 3.04 ± 0.17 13x 7.21 ± 0.86 3.29 ± 0.43 ^(a)Ca²⁺ channel agonist EC₅₀were determined by whole cell perforated patch clamp recordings ofdeactivation currents from N-type channels as described in the Methods.Cpd 13k was only soluble up to 200 μM in saline with 1% DMSO, thuslimiting our ability to accurately determine agonist properties.^(b)Cdk1 cyclinB(h) inhibitory EC₅₀ were determined in duplicate at 0.2,2, and 20 μM agent concentrations, with roscovitine as the positivecontrol. ND = not determined.

In addition to increasing Ca²⁺ channel agonist activity more stronglythan (R)-roscovitine, a compound with reduced cdk antagonist activitywas desired. A commercial kinase screen was used to test the effect ofthese novel compounds and (R)-roscovitine on several kinases, includingcdk1, cdk2, cdk5, MAPK1, and MLCK. FIG. 3C shows the dose response curveof cdk2 inhibition by the parent molecule and the three novel compounds.The IC₅₀ values for cdk2 activity following exposure to (R)-roscovitine,13w, 13u and 13x were 0.15±0.004 μM, 3.04±0.17 μM, 0.26±0.0002 μM and3.29±0.43 μM, respectively (Table 3) Against cdk1, cdk5, MAPK1 and MLCK,13x exhibited inhibitory activities EC₅₀=20.56±0.96, 3.03±0.32, >20,and >20 μM, respectively (Table 3), further illustrating a quitefavorable low-activity kinase profile for this structure. In light ofthe large cellular ATP concentrations (in the 1-10 mM range),single-digit μM activities of 13x against some kinases are likelyreadily compensated for in vivo and are therefore not consideredsignificant impediments from possible therapeutic applications of 13x asN/P/Q-type calcium channel agonist

TABLE 3 Comparison of (R)-roscovitine and analog EC₅₀/IC₅₀ affinities(in μM) for activity at calcium channels and kinases N-type P/Q-typeL-type Cdk1 Cdk2 Cdk5 MAPK MLCK (R)- 27.58 ± 1.65 120* >100^(†) 0.89 ±0.01  0.15 ± 0.004 0.14 ± 0.01 >20^(‡) >20^(‡) roscovitine 13w 30.02 ±1.87 N.D. N.D. 10.46 ± 2.77  3.04 ± 0.17 2.81 ± 0.91 >20^(‡) >20^(‡) 13u11.29 ± 1.48 N.D. N.D. 1.77 ± 0.04  0.26 ± 0.0002 0.27 ± 0.01 >20^(‡)19.45 ± 8.65 13x  7.21 ± 0.86 8.81 ± 1.07 >100^(†) >20^(‡) 3.29 ± 0.433.03 ± 0.32 >20^(‡) >20^(‡) *Literature EC₅₀ values for (R)-roscovitineon N- and P/Q-type Ca²⁺ channels taken from Buraei et al.(Neuropharmacology 2007, 52: 883) and Buraei and Elmslie (J. Neurochem2008, 105: 1450). The experimentally measured EC50 value for(R)-roscovitine on N-type channels was 27.58 ± 1.65 μM. ^(T)Nomeasureable agonist effect on L-type calcium channels up to 100 μM.^(‡)20 μM was the highest concentration used in kinase screens,therefore an IC₅₀ above 20 μM could not be reliably determined. N.D. =Not determined

Taken together, the data on Ca²⁺ channel and cdk activity show that 13xdisplays the most desirable properties of the compounds we havesynthesized and tested thus far, as it displays both a greatly increasedCa²⁺ channel agonist activity, and a decreased cdk2 antagonist activity,compared to the parent molecule (R)-roscovitine (Table 2). For thisreason, 13x was selected as the lead compound of interest.

The agonist activity of 13x on P/Q-type channels (Ca_(v) 2.1) and L-type(Ca_(v) 1.3) channels was evaluated using the same voltage-clampprotocol. Compound 13x had a very similar effect on P/Q-type channels asit did on N-type channels (EC₅₀=8.8±1.1 μM vs. 7.21±0.86 μM for P/Q- andN-type channels, respectively). Additionally, 13x increased the tailcurrent integral by ˜33-fold compared to control, similar to its effecton N-type channels (˜32-fold). Finally, 13x had no agonist activity(EC₅₀>100 μM) on the L-type α-subunit tested (Ca_(v) 1.3; Table 3).

In summary, 13x greatly improved upon (R)-roscovitine in terms of ourproperties of interest, with a ˜4-fold increase in efficacy as anagonist for N- and P/Q-type Ca²⁺ channels, a ˜3-4-fold increase inpotency as an agonist for N- and P/Q-type Ca²⁺ channels, and a ˜20-folddecrease in potency as a cdk antagonist (FIGS. 4A-4C).

For 13x, agonist effects on P/Q-type (EC₅₀=9.9 uM) and L-type calciumchannels (EC₅₀>100 uM) were evaluated. Error ranges for EC₅₀ values atN- and P/Q-type channels generated for 13x: 95% confidence interval fora fit to P/Q channels data=6.7-14.5 μM; 95% confidence interval for thefit to N-type channels=4.6-9.8 μM. Compound 13x was found to exhibitselectivity for N- and P/Q-type over L-type calcium channels.

EXAMPLE 2 Molecular Docking

Docking analyses to the cdk2/roscovitine complex were used to analyzepredictions for interactions with (R)-roscovitine analogs. Flexibledocking studies were performed using Molegro Virtual Docker (MVD,University of Aarhus, Denmark) to evaluate if the analogs bound to the(R)-roscovitine binding site of cdk2.²² The basis of these dockingstudies was a new hybrid search algorithm, i.e., a guided differentialevolution (DE) which combines DE optimization with a cavity predictionalgorithm which is dynamically used during the docking process. Briefly,all individual ligands were initialized, evaluated and scored(E_(score)/MolDock Score) according to the fitness function, which isthe sum of the intermolecular interaction energy between the ligand andthe protein and the intramolecular interaction energy of the ligand:E_(score)=E_(inter)+E_(intra), with E_(inter) being the ligand-proteininteraction energy and E_(intra) being the internal energy of theligand.

E_(PLP) is a piecewise linear potential using two different sets ofparameters: one set for approximating the steric van der Waals termbetween atoms and the other stronger potential for hydrogen bonds.E_(clash), assigns a penalty of 1000 if the distance between two atoms(more than two bonds apart) is less than 2.0 Å. Thus, the E_(clash) termpunishes infeasible ligand conformations.

Offspring were created using a weighted difference of the parentsolutions, which were randomly selected from the population. If, andonly if, the offspring was fitter, it replaced the parent. Otherwise,the parent survived and was passed on to the next generation,representing an iteration of the algorithm. The search process wasterminated when the number of fitness evaluations exceeded the maximumnumber of evaluations permitted.

Since the crystal structure of cdk2 (PDB ID: 3DDQ) contains(R)-roscovitine bound to the active site, it was possible to identifycritical residues surrounding the binding pocket. The MolDock scoringfunction in combination with the MolDock SE search algorithm and Tabuclustering²³ and a search space volume of 25 Å radius encompassing the(R)-roscovitine binding domain was chosen for docking, and both theligands and catalytic pocket residues were allowed to be flexible duringthe simulation. Each ligand was docked iteratively into the chosencavity in ten independent runs, each of which consisted of 1500 steps.Poses generated from each run were subjected to Tabu clustering wherebythe lowest energy pose below an energy threshold of 100 was generated asoutput. Thus, there were 10 poses per ligand ranked by energy. Thelowest energy pose of the 10 poses per ligand was selected for visualinspection.

For validation purposes docking was first applied to (R)-roscovitine,and the docked pose was computed to be very close to the position of theligand in the X-ray structure (i.e. RMSD=0.27 Å). The binding freeenergy, as estimated by the MolDock Score (arbitrary units) was −140,which indicated a strong interaction with cdk2 binding site. The sameprotocol was then applied to all (R)-roscovitine analogs.

The docking studies demonstrated that analogs 13d, 13g, 13k, 13w and 13xbound with a MolDock Score value between −127 and −143, and in adifferent orientation, less favorable than (R)-roscovitine. Compound 13ubound similarly to (R)-roscovitine with a MolDock Score of −142 (FIGS. 5and 6). These results were consistent with the experimental data thatshowed this compound to retain cdk2 activity, while 13d, 13g, 13k, 13wand 13x displayed reduced cdk2 activities. These results are consistentwith the experimental data that show this compound to retain cdk2activity, while 13d, 13g, 13k, 13w and 13x displayed reduced cdk2activities

EXAMPLE 3 Effects in LEMS Passive Transfer Model Mice

LEMS passive transfer. To test GV-58 in a LEMS model NMJ, an establishedLEMS passive transfer mouse model was utilized. To perform the passivetransfer of LEMS, mice were injected with the serum of patientsdiagnosed with LEMS. Collection of serum from LEMS patients wasperformed following the guidelines set forth by the University ofPittsburgh Institutional Review Board (IRB). Each serum sample wastested for the presence of voltage-gated Ca²⁺ channel antibodies using aCa²⁺ channel antibody radioimmune assay (Kronus RIA kit, Star, Id.).Control serum was obtained from the University of Pittsburgh MedicalCenter blood bank. Adult female CFW mice (2-3 months at beginning ofpassive transfer; 25-32 g; Charles River Laboratories) were divided intotwo groups: one group that received LEMS serum, and a control group thatreceived control serum. Mice received an intraperitoneal (i.p.)injection on day 1 of the treatment phase with 300 mg/kgcyclophosphamide to suppress immune responses, and were injected i.p.once per day for 30 consecutive days with either 1.5 mL serum from LEMSpatients or 1.5 mL control serum. In all cases, experimenters wereblinded to the injection conditions.

Intracellular recordings at mouse NMJs. Following the passive transferprotocol, intracellular recordings to assess the LEMS-mediated deficitin transmitter release were made in an ex vivo nerve-muscle preparation.A thin upper arm muscle, the epitrochleoanconeus (ETA), was chosen forthese recordings. This ex vivo nerve-muscle preparation was placed in abath containing 118 mM NaCl, 3.45 mM KCl, 11 mM dextrose, 26.2 mMNaHCO3, 1.7 mM NaH2PO4, 0.7 mM MgCl2, 2 mM CaCl2, pH=7.4. The nerve wasstimulated with a suction electrode and muscle contractions were blockedby exposure to 1 μM μ-conotoxin GIIIB (Alomone Labs). Microelectroderecordings were performed using ˜40-60 MΩ borosilicate electrodes filledwith 3 M potassium acetate. Spontaneous miniature synaptic events(mEPPs) were collected for 1-2 minutes in each muscle fiber, followed bysingle nerve evoked synaptic activity (10-30 EPPs) that was collectedwith an inter-stimulus interval of 10 seconds. A train of 10 EPPs wasalso collected in each muscle fiber using an inter-stimulus interval of20 msec. To analyze the data, both the amplitudes and the areas underthe waveforms (integral) were determined after correcting each digitizedpoint in each trace for non-linear summation. Data were collected andanalyzed using an Axoclamp 900A and the pClamp 10 suite of programs(Molecular Devices, Sunnyvale, Calif.).

Statistical Analysis. Statistical analysis was performed using eitherGraphPad Prism 5 (GraphPad Software, Inc.) or Origin 7 (OriginLab)software. For the dose-response analyses on Ca²⁺ current, eachconcentration of the four different compounds was tested in 3-6 cells.For the dose-response analyses on kinase activity, each of the threeconcentrations was tested in duplicates (n=2) for every compound except(R)-roscovitine, which was sent for kinase screening three times (n=6for each concentration). All EC₅₀ and IC₅₀ values and their respectiveerrors were determined by performing 100 iterations of a logisticfunction curve fit. Data are presented as mean±s.e.m. unless otherwisenoted.

Results. The effects of whole serum injections from 8 LEMS patients weretested by measuring the quantal content in mouse ETA neuromuscularjunctions following the passive transfer protocol (FIG. 7A), andcomparing them to the quantal content of mice that underwent a passivetransfer protocol with injections of normal human serum. The clinicalprofile for each LEMS patient whose serum was studied is shown in Table4. Several patients' serum caused no significant change in quantalcontent (FIG. 7A, black bars), whereas other patients' serum showedmoderate to strong changes in quantal content (FIG. 7A, white bars).

TABLE 4 Clinical data of each LEMS patient from whom serum was obtainedand tested. P/Q-type Ca²⁺ Age at CMAP ANNA1 channel Patient AgeDiagnosis increment (+/−) antibodies (+/−) PB 62 52  500% − + EB 66 57 331% − + PG 71 68 1300% − + JS 56 42  109% N.D. − aBC2 30 20800-1600% + + SH 61 55  78% + + LE 54 45  400% − + aCB 71 65  315% − +CMAP increment is a common diagnostic marker for LEMS and refers to theincrease in compound muscle action potential (CMAP) size following ashort exercise period (~10 seconds; Oh et al., 2007). ANNA1 =anti-neuronal nuclear antibody type I, which is also known as “anti-Hu”.N.D. = Not determined

In addition to testing quantal content following the passive transferprotocol, an antibody radioimmune assay was also performed to determinethe level of Ca²⁺ channel antibodies in each patient's serum (FIG. 7B).In general, those serum samples that significantly decreased quantalcontent had detectable levels of Ca²⁺ channel antibodies, although thelevel of these antibodies did not seem to correspond precisely to thelevel of quantal content decrease (FIGS. 7A and 7B).

The goal was to choose a single patient's serum for repeated testing ofthe novel calcium channel agonist (compound 13x) in order to have aconsistent passive transfer effect in every mouse. For these studies,the serum from patient aBC2 was selected because the quantal contentfollowing the passive transfer with this serum (40.5±9.9; mean±SD, n=49terminals) was significantly reduced compared to control serum(102.4±25.1; mean±SD, n=41 terminals, p<0.05, one-way ANOVA with Tukey'spost-hoc test; FIG. 7A, 7C). EPP amplitude following passive transferwith aBC2 serum was also significantly smaller than EPP amplitude ofNMJs injected with control serum (14.15±0.64 mV, n=49 vs. 34.61±1.37 mV,n=41 for aBC2 serum-treated NMJs and control serum-treated NMJs,respectively; p<0.05, Student's t-test), but mEPP amplitude was notsignificantly different between the two conditions (data not shown).Additionally, there was sufficient serum from this patient to performall of the desired experiments. Therefore, all of the following studieswere performed using mice that underwent the passive transfer protocolusing serum aBC2.

Compound 13x was tested in the LEMS model mice. Having developed aconsistent LEMS passive transfer protocol, the effect of compound 13x onaction potential-evoked transmitter release from LEMS passive transfermouse NMJs was evaluated. EPP amplitude and quantal content weredetermined in the vehicle (0.05%-0.1% DMSO) before a 30-minuteincubation in 50 μM 13x (FIG. 8A), which was then followed by repeatedEPP amplitude and quantal content measurements from the same NMJs withthe 13x still present in the bath. EPP amplitude was significantlyincreased from 13.00±0.56 mV (n=73 terminals) in vehicle-treated aBC2serum NMJs to 19.44±0.98 mV (n=73 terminals; p<0.05, Student's pairedt-test) following application of 50 μM 13x. The quantal content(determined by dividing the EPP peak amplitude by the mEPP peakamplitude) in the LEMS passive transfer vehicle control NMJs was38.0±12.8 (mean±SD, n=73 terminals), and was significantly increasedafter 13x exposure to 56.0±15.2 (mean±SD, n=73 terminals; p<0.05,Student's paired t-test; FIG. 8D). Furthermore, when the quantal contentwas determined from the area (integral) under EPP and mEPP waveforms,the quantal content in the vehicle controls was 38.3±12.7 (mean±SD, n=73terminals), and was significantly increased to 65.6±15.0 (mean±SD, n=73terminals; p<0.05, Student's paired t-test; FIG. 8E) following 13xapplication. The difference between the compound's effect on quantalcontent when measuring peak (˜62% increase) compared with its effect onquantal content when measuring area (˜92% increase) suggests that thereis a broadening of the EPP waveform caused by the action of 13x on Ca²⁺channels (expected based on the 13x-mediated slowing of Ca²⁺ currentdeactivation).

To further explore this possibility, both the full width at half maximum(FWHM) and the 90% to 10% decay time before and after 13x applicationwere measured. FIG. 8B shows an overlay of the average EPP amplitudes ina sample NMJ before (vehicle) and after 13x application. The FWHMincreased significantly from 3.39±0.06 ms in the vehicle controls (n=73terminals) to 3.90±0.07 ms following 50 μM 13x application (n=73terminals; p<0.05, Student's paired t-test). Similarly, the 90% to 10%decay time increased from 5.84±0.12 ms in vehicle controls (n=73terminals) to 6.79±0.11 ms following 13x application (n=73 terminals;p<0.05, Student's paired t-test). This indicates that the effect of 13xcannot fully be appreciated by only observing changes in peak EPPamplitude. Overall, 13x was shown to increase the strength ofneuromuscular transmission by about 50%. Because 13x increased the meanopen time of the calcium channels that trigger transmitter release, aslight broadening of the time-course of acetylcholine release resultedin a slight, but significant expansion of the postsynaptic potentials(FIG. 8B). These studies demonstrated that calcium channel agonists like13x can reverse neuromuscular weakness.

To ensure that the observed effect of 13x on transmitter release was notdue to inhibition of cdks, we tested the effect of olomoucine ontransmitter release at LEMS passive transfer mouse NMJs. Olomoucine is acompound that is structurally related to (R)-roscovitine and has potentcdk inhibitory activity, but no Ca²⁺ channel activity.

Application of 50 μM olomoucine caused a slight decrease in quantalcontent compared to vehicle controls when measuring quantal content frompeak (35.7±15.0, mean±SD, n=23 vs. 33.4±11.3, mean±SD, n=23, for vehiclecontrols and olomoucine, respectively; n<0.05, Student's paired t-test;FIG. 8D). The quantal content measured from area in vehicle controls(36.1±14.3, mean±SD, n=23) did not significantly change afterapplication of olomoucine (34.6±11.2, mean ±SD, n=23; p=0.16, Student'spaired t-test; FIG. 8E). Therefore, the effects of 13x on increasingaction potential-evoked transmitter release at LEMS passive transferNMJs appear to be due to effects on Ca²⁺ channels rather than effects oncdks.

In addition to analyzing the changes in quantal content and EPPkinetics, the effect of 13x on spontaneous transmitter release wasanalyzed. FIG. 8C shows sample mEPP traces recorded in the vehiclecontrol and following 50 uM 13x application. The mEPP frequency wassignificantly increased from 3.27±0.15 s⁻¹ (n=73) in vehicle controls to10.45±0.64 s⁻¹ (n=73) following application of 50 μM 13x (p<0.05,Students paired t-test). Furthermore, the mEPP amplitude did notsignificantly change following addition of 13x (mean change in amplitudefollowing 13x=1.00±0.02, n=73; p=0.86, Student's one sample t-test),thus confirming a presynaptic locus for effects.

Interestingly, some NMJs showed more than a 3-fold increase intransmitter release after exposure to 13x, while others showed a verysmall effect (see scatter plots in FIGS. 8D and 8E). There were severalpotential sources of variability in 13x effects on quantal content.First, during the relatively short (30-60 minutes) exposure, there mayhave been variable connective tissue barriers to diffusion, which mayhave resulted in different concentrations of 13x affecting particularNMJs within the muscle. It is also possible that the mix of calciumchannels at LEMS model synapses was variable when compared between NMJs(even in the same muscle). Compensatory changes in Ca²⁺ channelexpression have been reported to include an up-regulation of L-type Ca²⁺channel expression at the NMJ that might contribute to the triggering ofrelease at these disease model synapses, but L-type channels would notbe sensitive to modulation by 13x (see Table 3).

Finally, the effect of 13x on short-term plasticity was determined byeliciting a train of 10 stimuli at 50 Hz before and after application of50 μM 13x in the LEMS passive transfer model NMJs (FIGS. 9A, 9B). In thecontrol serum condition, there was almost no facilitation, and by the10^(th) EPP in the train there was a depression to about 66% of thefirst EPP. The trains of stimuli in the “aBC2” condition triggered EPPsthat were generally erratic in size during any single train, but theoverall average showed facilitation throughout the 50 Hz train, with apeak facilitation of ˜120% at EPP 4 and a small facilitation of ˜105%remaining at the final EPP in the train. When normalized to the firstEPP of the train, the control serum condition (n=41) was significantlydifferent than the “aBC2” condition (n=52) at each EPP in the trainfollowing the first (p<0.05, Student's t-test; FIGS. 9A, 9B).

The short-term plasticity characteristics before (0.05-0.1% DMSOvehicle) and after application of 50 μM 13x in the LEMS passive transfermodel NMJs were then compared (FIGS. 9C, 9D). The “Vehicle” conditionshowed facilitation throughout, with a facilitation of ˜113% remainingat the final EPP in the train. Following the application of 50 μM 13x inthe same NMJs, there was a slight facilitation followed by depression toabout 94% at the final EPP in the train. Furthermore, the “13x”condition was significantly different from the “Vehicle” condition atevery EPP following the first when both conditions were normalized tothe first EPP of the train (p<0.05, Student's paired t-test; FIGS. 9C,9D).

Representative traces in FIGS. 9A and 9C were chosen to display thedifferences in short-term plasticity characteristics rather than thedifferences in the first EPP's amplitude, which were quite variable (seeFIG. 8) among the four conditions. Asterisks in FIGS. 9B and 9D indicatea significant difference between the two normalized EPP amplitudes beloweach asterisk as determined by a Student's t-test in FIG. 9B or aStudent's paired t-test in FIG. 9D. Error bars indicate s.e.m.

Compound 13x increased the amount of Ca²⁺ influx through channels thatopen during an action potential, which in turn led to an increase in theamount of transmitter released (see, e.g., FIGS. 8A-8E). Whendetermining how such a use-dependent agonist would increase transmitterrelease at the mammalian NMJ, it is useful to consider thecalcium-dependent mechanisms that normally regulate release at thissynapse. The adult mouse NMJ has been shown to contain ˜850 very smallactive zones, each of which contains about two docked synaptic vesicles.Because the entire adult mouse ETA neuromuscular synapse releases about100 vesicles normally following each action potential stimulus (FIGS.7A-7C), the probability of release from each active zone is about 12%.Therefore, if each active zone only releases a synaptic vesicleapproximately 1 out of every 10 stimuli, the coupling between calciumchannel opening and vesicle fusion in these active zones may be verylow. Under these conditions, a use-dependent calcium channel agonistlike 13x would be expected to increase the flux through a subset of openchannels, increasing the probability of vesicle fusion at these sites.

One interesting observation was the lack of facilitation in the 50 Hztrain in control serum NMJs compared to the large facilitation presentin the 50 Hz train in LEMS serum-treated NMJs. If many Ca²⁺ channelscontribute to the release of a single vesicle within each active zone,as has been shown in multiple CNS synapses (then the large facilitationin the LEMS serum-treated NMJs would be caused by a smallerintracellular Ca²⁺ flux through fewer calcium channels at each activezone. Compound 13x would then compensate by increasing the Ca²⁺ influxthrough the remaining Ca²⁺ channels at the active zone. If, however, themouse NMJ functions as has been reported at the frog NMJ, there may be aroughly one-to-one relationship between Ca²⁺ channel opening and vesiclefusion. Under these conditions at the small, isolated active zonespresent at the mouse NMJ, an explanation for the increase infacilitation observed in the LEMS serum-treated NMJs is lessstraightforward. In this scenario, if the opening of one Ca²⁺ channelnormally contributes to the release of one vesicle (Ca²⁺ channel−releasesite cooperativity=1), then simply removing Ca²⁺ channels (as a resultof LEMS) should only reduce quantal content without affecting short-termplasticity since each release site that lost a calcium channel wouldsimply drop out, with no change in the calcium flux at release sitesthat had a calcium channel opening. On the other hand, if there is acompensatory expression of other types of Ca²⁺ channels in LEMS NMJs,this may result in the insertion of Ca²⁺ channels into sites furtheraway from the vesicle and its release machinery. This could lead to aCa²⁺-release site coupling such that it might be required that more thanone open Ca²⁺ channel provide the flux that is necessary for the releaseof a single vesicle. Under these conditions, one would predict anincreased facilitation during a 50 Hz train compared to control.Compound 13x would then reverse this by increasing the Ca²⁺ influxthrough each channel, thus increasing the likelihood that the fluxthrough a single channel could trigger the release of a synapticvesicle. Lastly, it is also possible that active zone structure andorganization is disrupted in the LEMS passive transfer NMJ. Disruptionof active zone structure and organization could alter the normallyone-to-one Ca²⁺ channel-to-vesicle coupling, thus accounting for boththe facilitation seen in the LEMS serum-treated NMJs and the partialrestoration of short-term plasticity characteristics by 13x as describedabove. LEMS could induce active zone disorganization in this scenario bydisrupting the interactions between Ca²⁺ channels and active zoneproteins following the autoimmune-mediated removal of Ca²⁺ channels. Forexample, previous work has shown that preventing the interaction betweenCa²⁺ channels and the active zone protein laminin p2 induces active zonedisorganization similar to that seen in LEMS NMJs.

EXAMPLE 4 Synergistic Action of DAP and 13x Completely ReversesNeuromuscular Weakness at LEMS Mode NMJs

The patch clamp data show that 13x has a greater effect when more Ca²⁺channels are open, which would occur when the depolarizing stimulus islonger in duration. This suggests the intriguing possibility that DAP(the current most common treatment for LEMS) and 13x would have asynergistic interaction because DAP opens more Ca²⁺ channels byprolonging the duration of the presynaptic action potential. To testthis, intracellular microelectrode recordings were performed on ex vivonerve-muscle preparations taken from LEMS passive-transfer model miceand measured the magnitude of acetylcholine released at theneuromuscular junction. The most sensitive method of quantifying themagnitude of acetylcholine released is to determine the quantal content.Quantal content is defined as the number of neurotransmitter-containingsynaptic vesicles that are released from the nerve terminal during asingle action potential stimulus. This value was determined by firstmeasuring the area under the average action potential-evoked endplatepotential (EPP) and then dividing this value by the area under theaverage single vesicle release event (miniature endplate potential;mEPP). This value could be compared between experimental groups toprovide an accurate measurement of changes in the magnitude ofacetylcholine released during a single action potential. Using thisapproach, the quantal content among five experimental conditions wascompared: control NMJs, LEMS NMJs, LEMS NMJs exposed to 50 μM 13x, LEMSNMJs exposed to 1.5 μM DAP, and LEMS NMJs exposed to 50 μM 13x+1.5 μMDAP (FIG. 10). A concentration of 1.5 μM DAP was used because previousstudies have reported that oral administration of DAP to patients leadsto peak serum levels of ˜70-150 ng/ml, which corresponds to aconcentration of ˜0.5-1.5 μM. First, NMJs taken from LEMS model miceshow significantly reduced quantal content (QC=26.7±1.4; EPPamplitude=10.18±0.62 mV) as compared with control serum treated mouseNMJs (QC=107.5±3.6; EPP amplitude=34.62±1.37 mV; Tarr et al., 2013).After exposure to 50 μM 13x, the quantal content in these LEMS NMJs wassignificantly larger (QC=48.4±2.7; EPP amplitude=13.75±1.244 mV). Infact, this 13x-mediated enhancement was very similar to what wasobserved after exposure of LEMS model NMJs to 1.5 μM DAP (QC=49.0±4.4;EPP amplitude=17.94±1.381 mV). Interestingly, when LEMS model NMJs wereexposed to a combination of 50 μM 13x plus 1.5 μM DAP, quantal contentincreased so much (QC=105.1±4.0; EPP amplitude=32.30±1.85 mV) that itwas not significantly different from the quantal content we measuredfrom NMJs taken from control serum-treated mice (QC=107.5±3.6; EPPamplitude=34.62±1.37 mV; Tarr et al., 2013). These data indicate thattransmitter release in LEMS model NMJs was completely restored tocontrol levels when exposed to both 13x and DAP (FIG. 10b ). In summary,while either 13x or DAP alone caused about an 80% increase intransmitter release from LEMS model NMJs, the combination of 13x plusDAP caused about a 300% increase in transmitter release.

In addition to measuring the properties of individual actionpotential-evoked events, the short-term plasticity characteristics amongall of the conditions by eliciting a train of 10 stimuli at 50 Hz wasalso measured and compared (FIG. 11). In NMJs taken from mice injectedwith control patient serum, the magnitude of transmitter release did notchange much during the first few stimuli in the train and depressedslightly to 66% of control levels by the 10^(th) stimulus in the train.In contrast, LEMS model NMJs showed strong facilitation throughout thetrain, with the 10^(th) EPP showing facilitation to ˜148% of the firstEPP. Both the 50 μM 13x condition (10^(th) EPP at ˜123% of the firstEPP) and the 1.5 μM DAP condition (10^(th) EPP at ˜132% of the firstEPP) showed only a partial restoration of short-term plasticitycharacteristics. However, when the combination of 50 μM 13x plus 1.5 μMDAP was given there was a near complete restoration of short-termplasticity characteristics, with a small amount of facilitation duringthe first few EPPs of the train and a depression at the 10^(th) EPP to˜73% of the first EPP (FIG. 11). The slight differences in short-termplasticity that persist in LEMS model NMJs treated with a combination of13x plus DAP may be due to several factors. First, even though thecombined effects of 13x and DAP can completely restore transmitterrelease magnitude in LEMS model NMJs, this occurs by enhancing both theprobability of opening (DAP) and the flux of calcium (13x) through thefewer than normal numbers of Ca²⁺ channels that remain in the activezone of these LEMS model NMJs. The enhanced Ca²⁺ flux at fewer than thenormal number of Ca²⁺ entry sites in these nerve terminals would bepredicted to create a different spatial and temporal profile ofpresynaptic Ca²⁺ concentration following each action potentialstimulation, which may enhance the residual calcium effects thatcritically influence short-term synaptic plasticity. Second, previousfreeze-fracture electron microscopic studies of LEMS active zones haverevealed a disruption in the organization of presynaptic proteins(presumed to include calcium channels. If this disruption changes thespatial distance between the remaining presynaptic calcium channels anddocked synaptic vesicles that are ready for release, this may alsoaffect short-term synaptic plasticity at these synapses.

Overall, the data show that exposure of LEMS model mouse NMJs to acombination of DAP plus the novel Ca²⁺ channel agonist (13x) completelyreverses the deficit in neurotransmitter release, which underlies theneuromuscular weakness that is characteristic of LEMS NMJs. This effectof the two compounds is not simply an additive effect, but rather asynergistic interaction, which is expected based on the mechanism ofaction of each compound.

-   Cell lines. For evaluation of effects of 13x on P/Q-type channels,    tsA-201 cells were transiently transfected with Ca_(v)2.1 in    combination with Ca_(v)β₃ and Ca_(v)α₂δ₁ (Addgene, Cambridge, Mass.)    using FuGENE 6 (Promega, Madison, Wis.). SH-SY5Y cells were used to    evaluate Cdk antagonist effects in the cell survival assay. All    cells were maintained in DMEM supplemented with 10% (tsA-201) or 15%    (SH-SY5Y) fetal bovine serum.-   Cell survival assay. A previously described, MTS-based cell survival    assay using SH-SY5Y cells was used to test Cdk antagonist effects in    the presence of physiological levels of ATP. The MTS reagent    (CellTiter 96® kit, Promega) is cell permeable and is reduced to a    colored product in viable cells that can be measured by the    absorbance at 490 nm. Briefly, SH-SY5Y cells were plated into    96-well clear-bottom plates. After 24 hours of drug treatment, the    MTS reagent was added and absorbance at 490 nm was determined using    an Infinite® Pro 200 microplate reader (Tecan). The absorbance    values in the drug-treated wells were normalized to the absorbance    values in wells containing the vehicle (0.05% DMSO). Background    absorbance was determined in wells containing no cells and was    subtracted from all values.-   Whole-cell perforated patch-clamp recordings. To assess the effects    of 13x, whole-cell currents through Ca²⁺ channels were recorded    using perforated patch methods as previously described (Tarr et al.,    2013). The pipette solution consisted of 70 mM Cs₂SO₄, 60 mM CsCl, 1    mM MgCl₂, 10 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid    (HEPES) at pH 7.4. The extracellular saline contained 130 mM choline    chloride (ChC1), 10 mM tetraethylammonium chloride (TEA-Cl), 2 mM    CaCl₂, 1 mM MgCl₂, 10 mM HEPES, at pH 7.4. Patch pipettes were    fabricated from borosilicate glass and pulled to a resistance of ˜1    MΩ. Capacitive currents and passive membrane responses to voltage    commands were subtracted from the data. A liquid junction potential    of −11.7 mV was subtracted during recordings. Currents were    amplified by an Axopatch 200B amplifier, filtered at 5 KHz, and    digitized at 10 KHz for subsequent analysis using pClamp 10 software    (Molecular Devices; Sunnyvale, Calif.). The tail current integral    was measured before and after application of a compound, with the    integral of each trace normalized to its peak. All experiments were    carried out at room temperature (22° C.). 13x was bath applied via a    glass pipette in a ˜0.5 mL static bath chamber during whole cell    recordings of calcium current. All other salts and chemicals were    obtained from Sigma-Aldrich (St. Louis, Mo.).-   LEMS passive transfer. To test 13x in a LEMS model NMJ, we utilized    an established LEMS passive transfer mouse model (Fukunaga et al.,    1983; Lang et al., 1984; Fukuoka et al., 1987; Smith et al., 1995;    Xu et al., 1998; Flink and Atchison, 2002). Collection of serum from    LEMS patients was performed following the guidelines set forth by    the University of Pittsburgh Institutional Review Board (IRB). Serum    from patient aBC2 was used for all studies reported here and was    collected using plasmapheresis. The serum was filtered with a 0.22 m    filter prior to the injection protocol. Adult female CFW mice (2-3    months old at beginning of passive transfer; weighing 25-32 g;    Charles River Laboratories, Wilmington, Mass.) received an    intraperitoneal (i.p.) injection on day 1 of the treatment phase    with 300 mg/kg cyclophosphamide to suppress immune responses, and    then were injected i.p. once per day for 15-30 consecutive days with    1.5 mL serum from LEMS patient aBC2. In all cases, experimenters    were blinded to the injection conditions.-   Intracellular recordings at mouse NMJs. Following the passive    transfer protocol, intracellular recordings to assess the    LEMS-mediated deficit in transmitter release were made in the mouse    epitrochleoanconeus (ETA) ex vivo nerve-muscle preparation in    accordance with procedures approved by the University of Pittsburgh    Institutional Animal Care and Use Committee as previously described    (Tarr et al., 2013). The extracellular saline contained 150 mM NaCl,    5 mM KCl, 11 mM dextrose, 10 mM HEPES, 1 mM MgCl₂, 2 mM CaCl₂,    pH=7.3-7.4. The nerve was stimulated with a suction electrode and    muscle contractions were blocked by exposure to 1 μM t-conotoxin    GIIIB (Alomone Labs, Jerusalem, Israel). Microelectrode recordings    were performed using ˜40-60 MΩ borosilicate electrodes filled with 3    M potassium acetate. To obtain the data required to calculate    quantal content, spontaneous miniature synaptic events (mEPPs) were    collected for 1-2 minutes in each muscle fiber, and then 10-30    nerve-evoked synaptic events (EPPs) were collected with an    inter-stimulus interval of 5 seconds. Each digitized point in each    trace was corrected for non-linear summation (McLachlan and Martin,    1981). To calculate quantal content, the integral of signal under    the average EPP was divided by the integral of signal under the    average mEPP recorded from each NMJ. This ratio calculates the    average number of quanta (packages of neurotransmitter stored in    synaptic vesicles) that are released following each presynaptic    action potential. To evaluate effects on short-term synaptic    plasticity, a train of 10 EPPs with an inter-stimulus interval of 20    msec (50 Hz) was collected in each muscle fiber. In some recordings    the protocol involved first performing vehicle (0.05% DMSO) control    recordings, then recording in the same muscle fibers after a 30-60    minute incubation in either 50 μM 13x or 1.5 μM DAP, and finishing    with recordings in the same muscle fibers again after a 30-60 minute    incubation in a combination of 50 μM 13x plus 1.5 μM DAP. In other    cases, we recorded from a group of muscle fibers in which we only    recorded vehicle controls before recording in the same muscle fibers    following a 30-60 minute incubation in a combination of 50 μM 13x    plus 1.5 μM DAP. Data were collected using an Axoclamp 900A and    digitized at 10 kHz for subsequent analysis using pClamp 10 software    (Molecular Devices, Sunnyvale, Calif.).-   Statistical analysis. Statistical analysis was performed using    either GraphPad Prism 5 (GraphPad Software, Inc., La Jolla, Calif.)    or Origin 7 (OriginLab Corporation, Northampton, Mass.). Data are    presented as mean±s.e.m. unless otherwise noted. A one-way ANOVA    with Tukey's post-hoc test was used to determine differences among    groups unless otherwise noted. The significance level was set at    p<0.05 for all tests.

EXAMPLE 5 Compound 13x is not Toxic to Cells Grown in Culture

Using a neuroblastoma cell line that is commonly used to evaluate cellcycle kinase inhibitors (SH-SY5Y cell line), when physiologicalconcentrations of ATP are present in cells, 13x (also referred to as“GV-58”) does not cause any effects on cyclin-dependent kinases orcreate any toxicity (while Roscovitine, the parent molecule, does). SeeFIG. 12.

In view of the many possible embodiments to which the principles of thedisclosed invention may be applied, it should be recognized that theillustrated embodiments are only preferred examples of the invention andshould not be taken as limiting the scope of the invention. Rather, thescope of the invention is defined by the following claims. We thereforeclaim as our invention all that comes within the scope and spirit ofthese claims.

We claim:
 1. A compound having a structure according to formula I or apharmaceutically acceptable salt thereof:

wherein each bond depicted as “------” is a single bond or a double bondas needed to satisfy valence requirements; Z¹, Z², Z³, Z⁴, and Z⁵independently are nitrogen or carbon; R¹ and R³ are alkyl; R² issubstituted or unsubstituted thiophenyl methyl; and R⁴ is alkyl orhydroxyalkyl.
 2. The compound of claim 1, wherein two of Z¹, Z², Z³, Z⁴,and Z⁵ are nitrogen.
 3. The compound of claim 1, wherein Z¹ and Z³ arenitrogen, and Z², Z⁴, and Z⁵ are carbon.
 4. The compound of claim 1,wherein R³ is ethyl.
 5. The compound of claim 1, wherein R⁴ is —CH₂OH.6. The compound of claim 1, wherein R¹ is n-alkyl.
 7. The compound ofclaim 1, wherein R¹ is C₁-C₃ alkyl.
 8. The compound of claim 7 where R¹is n-propyl.
 9. The compound of claim 1, wherein the structure is:


10. A pharmaceutical composition comprising: at least one compoundaccording to claim 1, or a pharmaceutically acceptable salt thereof; andat least one pharmaceutically acceptable additive.
 11. The compound ofclaim 1, wherein Z³ and Z⁴ are nitrogen, and Z¹, Z² and Z⁵ are carbon.12. The compound of claim 1, wherein R² is:


13. The compound of claim 2, wherein R² is:


14. The compound of claim 3, wherein R² is:


15. The compound of claim 1, wherein R¹ is substituted alkyl.
 16. Thecompound of claim 3, wherein R¹ is substituted alkyl.
 17. The compoundof claim 14, wherein R¹ is substituted alkyl.
 18. The compound of claim1, wherein R¹ is cyclic alkyl.
 19. The compound of claim 14, wherein R¹is cyclic alkyl.
 20. The compound of claim 11, wherein R¹ is substitutedalkyl.
 21. The compound of claim 1, wherein Z¹, Z² and Z³ are nitrogen,and Z⁴ and Z⁵ are carbon.
 22. The compound of claim 21, wherein R¹ issubstituted alkyl.